Production of encoded chemical libraries

ABSTRACT

This invention relates to the synthesis of nucleic acid-encoded chemical libraries using common adaptor sequences. Nucleic acid strands coupled to chemical moieties may be contacted with identifier oligonucleotides comprising coding sequences encoding the chemical moieties and an adaptor oligonucleotides, such that the adaptor oligonucleotide hybridizes to both the nucleic acid strands and the identifier oligonucleotides to allow ligation of the identifier oligonucleotides to the nucleic acid strands. The adaptor oligonucleotide is then removed. Nucleic acid-encoded chemical libraries, and methods of producing or screening such libraries are provided.

This invention relates to encoded chemical libraries, particularlynucleic acid-encoded self-assembling chemical libraries.

Nucleic acid-encoded chemical libraries are collections of chemicalmoieties covalently linked to identifier oligonucleotides encoding theidentity of the chemical moieties. The members of nucleic acid encodedchemical libraries display pharmacophores made up of one or morechemical moieties (also called “building blocks”). These chemicallibraries can be used to identify pharmacophores which are candidatebinding agents or have improved characteristics, for example improvedbinding.

Diverse populations of pharmacophores are produced by differentcombinations of chemical moieties. Each library member is tagged with anucleic acid strand comprising nucleotide sequences that encode thechemical moieties that constitute the pharmacophore that is displayed bythe member. This allows rapid identification of selected library membersduring screening.

DNA-encoded chemical library (DEL) technology allows the synthesis andscreening of pharmacophores of unprecedented size and quality. DELrepresents an advance in medicinal chemistry, which bridges the fieldsof combinatorial chemistry and molecular biology and promises torevolutionise the drug discovery field and to reshape the waypharmaceutically relevant compounds are traditionally discovered. Recentadvances in ultrahigh-throughput nucleic acid sequencing (e.g. Illuminasequencing, SOLiD technology, etc.) indicate that it should be possibleto sequence even billions of sequence tags per sequencing run. Withsuitable synthetic and encoding procedures, it should be possible toconstruct, perform selections, and decode nucleic acid-encoded librariescomprising multiple building blocks and containing millions of chemicalcompounds [1].

DNA encoded chemical libraries may display pharmacophores that areformed of chemically linked chemical moieties that are all attached to asingle strand of nucleic acid (“single pharmacophore libraries”) orpharmacophores that are formed of chemical moieties that are attached totwo different strands of nucleic acid hybridised together, one or morechemical moieties being attached to each strand (“dual pharmacophorelibraries”).

DNA-encoded chemical libraries were proposed first by Sydney Brenner andRichard Lerner in 1992 ([2]; U.S. Pat. No. 5,573,905; WO93/20242). Theseauthors postulated the alternating stepwise synthesis of a polymer (e.g.a peptide) and an oligonucleotide sequence (serving as a codingsequence) on a common linker (e.g. a bead) in split and pool cycles.After affinity capture on a target protein, the population of identifieroligonucleotides of the selected library members would be amplified byPCR and, in theory, utilised for enrichment of the bound molecules byserial hybridisation steps to a subset of the library. In principle, theaffinity-capture procedure could be repeated, possibly resulting in afurther enrichment of the active library members. Finally, thestructures of the chemical entities would be decoded by cloning andsequencing the PCR products. It was postulated that encoding procedurescould be implemented by a variety of methods, including chemicalsynthesis, DNA polymerization or ligation of DNA fragments [2].

The feasibility of the orthogonal, solid-phase synthesis of peptides andoligonucleotides was demonstrated by attaching a test peptide (thepentapeptide leucine enkephalin) and an encoding identifieroligonucleotide onto controlled-pore glass beads [3]. The peptide boundto a specific antibody and the corresponding DNA coding tag wasamplified by PCR. The technology was used to construct a collection of˜10⁶ heptapeptide sequences and their corresponding identifieroligonucleotide tags on beads. The library was incubated with afluorescently labelled anti-peptide antibody, and binders were selectedsuccessfully by fluorescent-assisted cell sorting [4]. In their originalpaper, Brenner and Lerner suggested that the alternate synthesis ofchemical compounds and oligonucleotides could also be implemented in theabsence of beads. The use of enzyme catalysed ligation of coding DNAfragments is now established in the field (US 2006-0246450; WO02/103008; WO2004/013070; WO2004/074429; WO2007/062664 andWO2005/058479).

Various methods of generating DNA-encoded chemical libraries have beendescribed in the art (see for example [1, 2, 5-9]; WO2009/077173;WO2003/076943)

Standard strategies for encoding DNA-encoded chemical libraries based ontwo sets of building blocks involve the stepwise ligation ofdouble-stranded DNA-fragments (containing a code for the unambiguousidentification of each building block) after each addition of a chemicalmoiety in the library ([2], US 2006-0246450; WO 02/103008;WO2004/013070; WO2004/074429; WO2007/062664 and WO 2005/058479). Inother words, the insertion of a chemical building block in a nascentchemical structure is associated with the ligation of a DNA-fragmentthat serves as code for that building block. The final code of thecomplete molecule is provided by the sum of the codes, corresponding tothe individual building blocks.

However, in these standard strategies, each building block requires thesynthesis and subsequent ligation of two complementary oligonucleotides.Thus, the synthesis of a library with n×m building blocks requires thesynthesis of 2n+2m oligonucleotides.

A more economical method for the encoding of DNA-encoded chemicallibraries consisting of two sets of n×m building blocks involvescoupling the first n chemical moieties at the 5′ end of noligonucleotides [1]. In a split and pool synthetic strategy, theaddition of m second building blocks to the nascent chemical structureswas encoded by annealing the first n oligonucleotides with m partiallycomplementary oligonucleotides that provided a code for the secondreaction step. The structures were converted into double-strandednucleic acid fragments by a Klenow-assisted polymerization step.Although this method is simple and efficient, it is not applicable forthe synthesis of libraries with 3 or more sets of building blocks.

A general and economical method for the encoding of nucleic acid-encodedchemical libraries, based on any number of sets of building blocks wouldtherefore be useful.

The present inventors have recognised that the annealing ofoligonucleotides with common adaptor sequences allows the efficient andeconomical synthesis of nucleic acid-encoded chemical libraries. Forexample, a nucleic acid-encoded chemical library or a sub-library foruse in generating a nucleic acid-encoded chemical library may besynthesised using fewer than two oligonucleotides for the addition andencoding of each building block.

A first aspect of the invention provides a method of producing a nucleicacid encoded chemical sub-library comprising;

-   -   (i) providing a population of nucleic acid strands, each nucleic        acid strand being coupled or couplable to one or more members of        a diverse population of chemical moieties,    -   (ii) contacting the nucleic acid strands coupled or couplable to        the one or more chemical moieties with identifier        oligonucleotides comprising coding sequences and one or more        adaptor oligonucleotides, such that the adaptor oligonucleotides        hybridize to both the nucleic acid strands and the identifier        oligonucleotides to form partially double-stranded trimeric        complexes,    -   wherein each nucleic acid strand is contacted with an identifier        oligonucleotide comprising a coding sequence that encodes a        chemical moiety that is coupled or couplable to the nucleic acid        strand, and;    -   wherein each adaptor oligonucleotide forms multiple complexes        with different nucleic acid strands and identifier        oligonucleotides,    -   (iii) ligating the nucleic acid strands to the identifier        oligonucleotides in the partially double-stranded complexes,        such that the identifier oligonucleotides are incorporated into        the nucleic acid strands,    -   thereby producing a sub-library of chemical moieties coupled to        nucleic acid strands, wherein one or more chemical moieties are        coupled to each nucleic acid strand and each nucleic acid strand        comprises a coding sequence that encodes a chemical moiety        coupled to the nucleic acid strand.

Preferably, all the nucleic acid strands in the population are contactedwith the same adaptor oligonucleotide i.e. the same adaptoroligonucleotide hybridises to all of the nucleic acid strands in thepopulation and all of the identifier oligonucleotides.

A nucleic acid-encoded self-assembling library or sub-library is acollection of library members or nascent library members, each of whichdisplays a pharmacophore that is made up of one or more chemicalmoieties. The identity of the one or more chemical moieties thatconstitute the pharmacophore is encoded into each library member througha nucleic acid strand that incorporates a coding sequence. The membersof the library display a diverse population of pharmacophores. Thisallows the screening of a large number of pharmacophores. For example, anucleic acid-encoded self-assembling library may comprise 10⁶ or moredifferent pharmacophores for screening.

Two or more nucleic acid encoded sub-libraries may be combined togenerate a nucleic acid-encoded self-assembling library.

Nucleic acid-encoded libraries may be useful in the identification ofpharmacophores which are candidates for binding to a target of interest,such as a protein, or which have improved characteristics compared topreviously known pharmacophores, such as improved binding affinity to atarget of interest. Suitable targets for nucleic acid-encoded librariesof pharmacophores are well known in the art.

Following screening, the identity of the pharmacophore that is displayedby a selected library member may be determined by decoding the codingsequences that are incorporated into the nucleic acid strand of theselected library member.

A member of a nucleic acid-encoded chemical library (‘library member’)may be formed from two nucleic acid strands (a first and a secondstrand) and one or more chemical moieties.

A nucleic acid strand may be DNA, RNA or chimeric RNA/DNA. Preferably,the nucleic acid strand(s) in the chemical libraries described hereinare DNA.

The first nucleic acid strand of the library member may be coupled toone, two, three, or more than three chemical moieties and the secondnucleic acid strand of the member may be coupled to zero, one, two,three, or more than three chemical moieties. The chemical moieties maybe coupled to one of the 5′ and 3′ ends of the first nucleic acid strandand the other of the 5′ and 3′ ends of the second nucleic acid strand.The chemical moieties in each member form the pharmacophore that isdisplayed by the member. A chemical library contains library membersthat together display a diverse population of pharmacophores. Thenucleic acid strands hybridize to form a duplex nucleic acid moleculewhich is coupled to the pharmacophore that is displayed by the librarymember. The nucleic acid strands may self-assemble through thehybridization of complementary regions in each strand to form adouble-stranded or partially double stranded nucleic acid molecule.

In some embodiments, a chemical library member may be formed from afirst nucleic acid strand coupled to one or more chemical moieties thatform a pharmacophore, and a second nucleic acid strand that ishybridised to the nucleic acid strand that is not coupled to a chemicalmoiety (i.e. only the first nucleic acid strand contributes to thepharmacophore). For example, the first nucleic acid strand may becoupled to three chemical moieties and the second strand may not becoupled to any chemical moieties.

In other embodiments, a chemical library member may be formed from afirst nucleic acid strand and a second nucleic acid strand, each ofwhich is coupled to one or more chemical moieties, such that thechemical moieties coupled to the strands together form a pharmacophore(i.e. both the nucleic acid strands contribute to the pharmacophore).For example, the first nucleic acid strand may be coupled to onechemical moiety and the second nucleic acid strand may be coupled to onechemical moiety or the first nucleic acid strand may be coupled to twochemical moieties and the second nucleic acid strand may be coupled toone chemical moiety.

The nucleic acid strands that form the library members may themselves bemembers of a sub-library, each nucleic acid strand in the sub-librarybeing coupled to a different chemical moiety or combination of chemicalmoieties.

The self-assembled library may be formed from the hybridisation of asub-library of first nucleic acid strands, each first nucleic acidstrand being coupled to one or more members of a first diversepopulation of chemical moieties, with a sub-library of second nucleicacid strands, each second nucleic acid strand being coupled to one ormore members of a second diverse population of chemical moieties. Thefirst and second diverse populations may be the same or different. Whenthe sub-libraries of nucleic acid strands hybridise together to formdouble-stranded library members, pharmacophores are generated from thedifferent combinations of the chemical moieties coupled to the nucleicacid strands. This increases the number of different pharmacophores inthe library.

A sub-library may comprise different chemical moieties coupled tonucleic acid strands. The nucleic acid strands in a sub-library mayassemble through hybridisation with nucleic acid strands from the samesub-library or a different sub-library, for example a sub-library ofnucleic acid strands conjugated to a different number of chemicalmoieties, to produce a double-stranded library. For example, in thesimplest scenario, a sub-library comprising nucleic acid strands coupledto one chemical moiety may assemble with a partner nucleic acid strandwhich is not coupled to a chemical moiety. The pharmacophore would thenconsist of one chemical moiety. In a further example, nucleic acidstrands coupled to single chemical moieties may assemble with nucleicacid strands coupled to two chemical moieties, thereby presenting apharmacophore consisting of three chemical moieties. These examples arefor illustration only and any number of combinations can be envisaged.For some purposes, a pharmacophore formed by chemical moieties coupledto one nucleic acid strand may be preferred as the moieties are boundtogether and therefore represent a lead-like compound. For otherpurposes, a pharmacophore formed by chemical moieties coupled todifferent nucleic acid strands may be preferred.

A nucleic acid strand is a polynucleotide chain (e.g. a DNA, RNA orRNA/DNA chain) which may be coupled to one or more chemical moieties. Anucleic acid strand may hybridize to a second nucleic acid strandthrough complementary regions in the two strands. A nucleic acid strandmay be coupled to 0, 1, 2, 3, 4, 5 or more chemical moieties which mayform all or part of the pharmacophore.

When a nucleic acid strand hybridizes to one or more partner nucleicacid strands to form a library member, the chemical moieties that arecoupled to the strands form the pharmacophore that is displayed by thelibrary member. In some embodiments, only one of the strands is coupledto chemical moieties in the pharmacophore (often termed a“single-pharmacophore” library). In other embodiments, both of thestrands may be coupled to chemical moieties in the pharmacophore (oftentermed a “dual-pharmacophore” library).

During the production of a library member as described herein, a nucleicacid strand may act as a scaffold into which one or more codingsequences are incorporated, for example by the ligation of identifieroligonucleotides comprising coding sequences onto the nucleic acidstrand or the polymerase mediated extension of the nucleic acid strandalong a template that comprises the complement of a coding sequence.

In some embodiments, a nucleic acid strand for use in the methodsdescribed herein may initially contain coding sequences encoding one ormore chemical moieties. For example, the nucleic acid strand mayinitially contain a coding sequence that encodes the chemical moietythat is coupled or couplable to the nucleic acid strand. One or morefurther identifier oligonucleotides comprising coding sequences encodingfurther chemical moieties coupled or couplable (i.e. capable of beingcoupled) to the nucleic acid strand may be added during the productionof the library member as described herein.

A nucleic acid strand may initially be coupled to one or more chemicalmoieties and further chemical moieties may be subsequently coupled tothe nucleic acid strand.

The nucleic acid strand may be coupled to the chemical moiety at anystep of the methods disclosed herein. For example, the nucleic acidstrand may be coupled to the chemical moiety before, or after the stepof ligating the nucleic acid strand to the identifier oligonucleotide.For example coupling may be performed before step (i), before step (ii)or after step (iii). Suitable methods for coupling chemical moieties tonucleic acid strands are well-known in the art.

The nucleic acid strand may comprise a proximal end that is coupled tothe chemical moiety, for example the 5′ end, and a distal end to whichthe coding sequence is added, for example the 3′ end. An annealingregion which hybridises with the adaptor oligonucleotide may be locatedadjacent the distal end of the nucleic acid strand to facilitateligation of an identifier oligonucleotide.

The nucleic acid strand may comprise first and second hybridisationregions to anneal primers for the amplification of the strand, forexample after screening. Amplification products produced byamplification of the nucleic acid strand may comprise the codingsequences encoding the chemical moieties that form the pharmacophoredisplayed by the library member. The typical length of regions forprimer annealing is between 10 and 28 nucleotides (in single strandedformat), or base pairs (in double stranded format). In double strandedformat, one of the two nucleic acid strands of the region may form asequence specific dimer with a PCR primer at an appropriatehybridisation temperature. A typical hybridisation temperature for thesequence specific hybridisation of PCR primers to PCR primer regions isbetween 40 and 70° C. PCR primers can be longer than the hybridisationregion of the nucleic acid strand or contain additional sequences (e.g.,at their respective 5′ ends). This may be useful for later steps of thedecoding process.

The one or more chemical moieties coupled to the nucleic acid strand orstrands of a library member form a pharmacophore. A pharmacophore is acollection of molecular features or elements which is capable ofspecifically interacting with a target. Different combinations ofchemical moieties produce different pharmacophores which are displayedby different members of the library.

The pharmacophore may be formed from chemical moieties which arecovalently bound together; chemical moieties which are not covalentlybound together; or combinations of both. Typically, chemical moieties onthe same nucleic acid strand will be covalently bonded together andchemical moieties on different nucleic acid strands will be broughttogether by the assembly of the nucleic acid strands. For example, oneor more chemical moieties coupled to a nucleic acid strand may associatewith one or more moieties attached to a partner nucleic acid strand toform a pharmacophore.

Suitable targets for binding to pharmacophores include biologicaltargets, for example biological macromolecules, such as proteins. Thetarget may be a receptor, enzyme, antigen or oligosaccharide. The targetmay be a compound, for example a synthetic compound. The interactionwith the target is generally through specific binding of all or part ofthe pharmacophore with the target. In other words, some or all or thechemical moieties, or parts of the chemical moieties which form thepharmacophore may specifically bind to the target.

The binding between the pharmacophore and target may occur throughintermolecular forces such as ionic bonds, hydrogen bonds and van derWaals forces, which are generally reversible. The binding may occurthrough covalent bonding, which is generally irreversible, although thisis generally rare in biological systems.

The pharmacophore formed by the chemical moieties may, for example, be aligand, substrate, inhibitor or activator or may be useful in thedevelopment thereof. The pharmacophore may be an agonist or antagonistor a candidate agonist or antagonist or may be used as a model or leadin the development of such an agonist or antagonist.

A chemical moiety may form all or part of a pharmacophore. A singlechemical moiety may be the pharmacophore or preferably may associatewith other chemical moieties coupled to the library member to form apharmacophore comprising multiple chemical moieties. The pharmacophoreand/or chemical moieties are used in screening.

A chemical library member may display a pharmacophore which comprises orconsists of any of 1, 2, 3, 4, 5 or more chemical moieties. The chemicalmoieties may be attached to one or both nucleic acid strands. Forexample, a first strand may be coupled to 1, 2, 3, 4, 5 or more chemicalmoieties and a second strand may be coupled to 0, 1, 2, 3, 4, 5 or morechemical moieties

In some preferred embodiments, the total molecular weight of thechemical moieties in the pharmacophore may be less than 1 kD, preferablyless than 500 D.

Suitable chemical moieties include small organic molecules, amino acidresidues or other amino-containing moieties (optionally with appropriateamino protection); and peptides or globular proteins (including antibodydomains). In some embodiments, a chemical moiety may have a molecularweight of 300 Da or less, for example about 100 to 300 Da. Populationsof chemical moieties for use in the generation of libraries are wellknown in the art (see [1] to [9]).

A chemical moiety may be covalently coupled to the nucleic acid stranddirectly or indirectly, for example via a linker. Suitable linkers, suchas alkyl chains, are well known in the art. The chemical moiety may becoupled directly using conventional synthetic chemistries, for exampleamide or other conventional linkages.

Chemical moieties may be coupled to a nucleic acid strand via otherchemical moieties. For example, each of the chemical moieties coupled toa nucleic acid strand may be covalently bonded to other chemicalmoieties and one of the chemical moieties may be coupled to the nucleicacid strand. Suitable methods for covalently bonding chemical moietiesare well known in the art. In some embodiments, a pharmacophore may beformed from a single compound comprising the covalently-bound chemicalmoieties coupled to a nucleic acid strand.

In other embodiments, the pharmacophore displayed by a library membermay be formed from two or more chemical moieties which are covalentlybonded to each other, and a further one or more chemical moieties whichare not covalently bonded to the covalently bonded moieties.

For example, two or more covalently bonded chemical moieties may becoupled to one of a nucleic acid strand or a partner nucleic acid strandand form a pharmacophore with a further one or more chemical moietiescoupled to the other of a nucleic acid strand or a partner nucleic acidstrand, when the strands assemble to form a double-stranded librarymember.

An example of this structure is shown in FIGS. 2A, 2B and 2C, where thepharmacophore is formed from bbA, bbB and bbC. bbB and bbA arecovalently bonded to each other and are also coupled to a nucleic acidstrand while bbC is coupled to a partner nucleic acid strand but is notcovalently bonded to bbA or bbB.

A chemical moiety may be coupled to the 5′ or 3′ terminal of a nucleicacid strand.

If there are two or more chemical moieties coupled to a nucleic acidstrand, these may be joined to one another by one or more chemicalreactions and their residues will be linked by one or more chemicalbonds.

The chemical moieties may be joined to one another by covalent bonds orby non-covalent interactions.

Library members as described herein comprise a double-stranded nucleicacid molecule. Preferably, one of the nucleic acid strands comprisescoding sequences that encode all of the chemical moieties thatconstitute the pharmacophore displayed by the library member.

The adaptor oligonucleotide serves as a template to facilitate theligation of the nucleic acid strand and the identifier oligonucleotidecontaining the coding sequence. A single adaptor oligonucleotide mayfacilitate the ligation of multiple nucleic acid strands and identifieroligonucleotides. For example, a set consisting of 1, 2, 3, 4, 5 or moreadaptor oligonucleotides may be used to facilitate ligation of all ofthe nucleic acid strands in the population to the identifieroligonucleotide containing the corresponding coding sequence.Preferably, the sequence of the adaptor oligonucleotide is the sameregardless of the chemical moiety(s) coupled to the nucleic acid strandi.e. only 1 adaptor oligonucleotide is used. This reduces the totalnumber of oligonucleotides required to generate the nucleic acid encodedchemical library.

The adaptor oligonucleotide hybridises with the nucleic acid strand andidentifier oligonucleotide and brings the ends of the nucleic acidstrand and identifier oligonucleotide into association within adouble-stranded trimeric complex, such that they can be ligated togetherby a ligase. The adaptor may bring into association the 3′ end of thenucleic acid strand to the 5′ end of the identifier oligonucleotide orthe 5′ end of the nucleic acid strand to the 3′ end of the identifieroligonucleotide.

The association is maintained by hybridization of a first annealingregion of the adaptor to a complementary annealing region of the nucleicacid strand and a second annealing region of the adaptor to acomplementary annealing region of the identifier oligonucleotide. Thecomplementary annealing regions contain a nucleotide sequence that iscomplementary to the annealing region. The sequences of thecomplementary annealing region of the nucleic acid strand and theidentifier oligonucleotide are the same, regardless of the chemicalmoiety(s) coupled to the nucleic acid strand, to allow the same adaptoroligonucleotide sequence to be used to encode all the chemical moietiesin the library.

The first annealing region of the adaptor may be proximal to thechemical moiety and may hybridise with a complementary annealing regionat the distal end of the nucleic acid strand. The second annealingregion of the adaptor may be distal to the chemical moiety and mayhybridise with the proximal complementary annealing region of theidentifier oligonucleotide.

The proximal and distal annealing regions of the adaptor may comprise orconsist of nucleic acid or RNA bases or both. The proximal and distalannealing regions contain sufficient numbers of bases so that they canhybridise to complementary regions on the nucleic acid strand andidentifier oligonucleotide respectively. Typically, an annealing regionwill contain at least 6 nucleotides, and more preferably at least 9nucleotides. Normally, the annealing region will contain no more than 20nucleotides, preferably not more than 15 nucleotides. For example, thefirst and second annealing regions of the adaptor may be 9 to 15 basesin length. A suitable adaptor oligonucleotide may, for example be 15 to35 bases, preferably 18 to 30 bases in length.

The adaptor oligonucleotide hybridises to the nucleic acid strand andthe identifier oligonucleotide such that a complex that comprises adouble-stranded region is formed in the vicinity of the ends of thenucleic acid strand and the identifier oligonucleotide that are to beligated.

In some embodiments, the adaptor may remain hybridised to the firstnucleic acid strand, for example within a nucleic acid spacer strand,and may form part of the library member that is produced.

In other embodiments, the adaptor may be removable or removed bypurification following the ligation step. For example, adaptor may beseparated under denaturing conditions on the basis of their small sizerelative to the nucleic acid strand incorporating the identifieroligonucleotide.

More preferably, the adaptor oligonucleotide may be cleavable. Cleavageof the adaptor oligonucleotide produces fragments which are too short toremain hybridised to the nucleic acid strand and the identifieroligonucleotide. The adaptor oligonucleotide is thus removed from thenucleic acid strand and the identifier oligonucleotide by cleavage afterligation has occurred.

Cleavage or degradation of the adaptor results in separation of theadaptor oligonucleotide from the nucleic acid strand. In someembodiments, the nucleic acid strand may be purified following removalof the adaptor for example to remove fragments of a cleaved or degradedadaptor. Suitable purification methods are well known in the art.

The adaptor oligonucleotide may be cleaved enzymatically, for exampleusing RNAase, or chemically, for example by base hydrolysis (typically,exposure to pH>12 at room temperature or greater).

The adaptor oligonucleotide may be DNA, RNA or chimeric (i.e. containingboth deoxyribonucleotides and ribonucleotides).

In some preferred embodiments, the adaptor oligonucleotide is chimeric.In addition to deoxyribonucleotides, a suitable chimeric adaptoroligonucleotide may comprise one or more ribonucleotides, for exampletwo or more, three or more, four or more or five or moreribonucleotides. The ribonucleotide bases in the adaptor may beconsecutive or non-consecutive. The ribonucleotide sequence may belocated within the adaptor such that cleavage of the RNA, e.g. viahydrolysis produces fragments which are too short to hybridise to thenucleic acid strand and the identifier oligonucleotide. For example, theadaptor may comprise one or more contiguous sequences of 2, 3, 4, 5, 6,7, 8, or more deoxyribonucleotide bases that are separated by 1, 2, 3, 4or more ribonucleotide bases. Examples of suitable chimeric adaptoroligonucleotides for use as described herein are shown in Tables 1 and2.

RNA adaptors or chimeric adaptor oligonucleotides may be convenientlycleaved by treatment with RNAase. RNAse is a nuclease which catalysesthe degradation of RNA into smaller components. RNAse is readilyavailable from commercial suppliers.

Suitable adaptor oligonucleotides may be synthesised using appropriatetechniques.

Hybridization of the adaptor oligonucleotide to the nucleic acid strandand the identifier oligonucleotide brings an end of the nucleic acidstrand into proximity to an end of the identifier oligonucleotide sothat a ligase enzyme may act to join the identifier oligonucleotide tothe nucleic acid strand. Suitable hybridisation conditions for thehybridisation of polynucleotides are well-known in the art and includefor example a temperature of between 0° C. and 70° C.

Identifier oligonucleotides suitable for use as described hereincomprise a coding sequence that encodes a chemical moiety.

The coding sequence (or coding region) can be any sequence of nucleicacid bases that is uniquely associated with a particular chemicalmoiety. This allows the identity of the chemical moiety to be determinedby sequencing or otherwise ‘reading’ the coding sequence.

A coding sequence contains sufficient nucleotides to uniquely identifythe chemical moiety for which it is coding. For example, if the chemicalmoiety has 20 variants, the coding sequence needs to contain at least 3nucleotides (4²=16, 4³=64). The coding sequence may be longer thannecessary. The benefit of employing coding sequences that are longerthan necessary is that they provide the opportunity to differentiatecodes by more than just a single nucleotide difference, which gives moreconfidence in the decoding process. For example, a first chemical moietyfrom a population of 20 different moieties (20 compounds) may be encodedby 6 nucleotides, and a second chemical moiety from a population of 200different moieties may be encoded by 8 nucleotides. The size of thecoding sequence therefore depends on the number of chemical moieties tobe encoded (i.e. the number of different chemical moieties in thelibrary). A sequence of nucleotides and/or its complement may be used asa coding sequence to encode a chemical moiety. Suitable sequences forencoding chemical moieties in a library are well-known in the art.

Preferably, the coding sequences of the identifier oligonucleotides areflanked by constant regions. The constant regions are of sufficientlength to allow an efficient hybridization and ligation, for example3-20 bases, preferably 9-15 bases. Examples of suitable sequences areshown in Tables 5-7.

The constant regions of the identifier oligonucleotide may comprise oneor more complementary annealing regions that hybridize to annealingregions of the adaptor. Preferably, the sequence of the complementaryannealing region is the same in all the identifier oligonucleotidesregardless of the coding sequence i.e. identifier oligonucleotidesencode the different chemical moieties (and therefore having differentcoding sequences) comprise the same complementary annealing region. Thisallows a single adaptor to be used for the ligation of differentidentifier oligonucleotides to nucleic acid strands.

The complementary annealing regions of the identifier oligonucleotideare of sufficient length to allow for specific hybridisation between thecomplementary annealing region and the annealing region of the adaptor.Typically, a complementary annealing region will contain at least 6nucleotides, more preferably at least 9 nucleotides and no more than 15nucleotides. Suitable conditions for the sequence specific hybridisationof two polynucleotides are well known in the art.

Complementary annealing regions may be located either side of the codingsequence in the identifier oligonucleotide. For example, a firstcomplementary annealing region may be on the proximal side of the codingsequence (i.e. nearest the end that is ligated to the nucleic acidstrand) and a second complementary annealing region may be on the distalside of the coding sequence (i.e. furthest the end that is ligated tothe nucleic acid strand). The first and second complementary annealingregions may have the same sequence or more preferably differentsequences to each other.

The complementary annealing region may be capable of hybridizing to acomplementary region in a second nucleic acid strand to facilitate theassembly of double-stranded library members.

Identifier oligonucleotides and chemical moieties may be added to thelibrary member in alternate cycles, at least partly because of differentreaction conditions being required for the two steps. In someembodiments, the chemical moiety can be added first and then itsidentifier oligonucleotide incorporated into the nucleic acid strand, orthe identifier oligonucleotide can be incorporate into the nucleic acidstrand and then the chemical moiety encoded by the incorporatedidentifier oligonucleotide may be added to the nascent library member.In methods of the invention, coupling may occur before step i) beforestep ii) or after step iii). In some cases, the identifieroligonucleotide may be used to direct synthesis or addition of theencoded chemical moiety, in which case the identifier oligonucleotidemay be added before the chemical moiety.

The adaptor may be hybridized to the nucleic acid strand and identifieroligonucleotide through the annealing of annealing regions in theadaptor to complementary annealing regions in the nucleic acid strandand the identifier oligonucleotide.

Hybridization establishes a non-covalent sequence-specific base-pairingbetween one or more complementary strands of nucleic acids. Undersuitable reaction conditions the complementary strands will anneal andform a double stranded complex. Suitable hybridisation conditions arewell-known in the art. Typical hybridisation temperatures for thesequence specific annealing of two polynucleotide strands may be between0° C. and 70° C.

The nucleic acid strand may be ligated to the identifier oligonucleotideby any suitable technique known in the art.

Preferably, the nucleic acid strand and identifier oligonucleotide maybe enzymatically ligated, e.g. using DNA or RNA ligase. DNA and RNAligases catalyze the formation of a phosphodiester bond between the 3′hydroxyl and 5′ phosphate of adjacent DNA or RNA residues, respectively.The ligation step joins an end of the nucleic acid strand to an end ofthe identifier oligonucleotide such that the identifier oligonucleotideis incorporated into the nucleic acid strand. Suitable ligationconditions are well-known in the art.

In some embodiments, following ligation, the adaptor may be separated orremoved from the nucleic acid strand comprising the ligated identifieroligonucleotide, for example by cleavage or degradation, as describedabove. In other embodiments, the adaptor may remain hybridised as partof the double stranded nucleic acid molecule within library members.

The nucleic acid strand may be hybridised to a second nucleic acidstrand in order to assemble a library member comprising adouble-stranded nucleic acid molecule.

The second nucleic acid strand is capable of hybridizing to the nucleicacid strand, as described above. The second nucleic acid strand maycomprise one or more hybridization regions which hybridize tocomplementary regions in the first nucleic acid strand thereby allowingthe first and second nucleic acid strands to assemble or dimerise as adouble-stranded or partially double stranded complex. Suitablehybridization regions may comprise between 18 and 24 bases, in order forthe nucleic acid strands to self-assemble into a library member.

One or both of the nucleic acid strands may comprise first and secondprimer regions as described above.

As described above, the second nucleic acid strand may be coupled to oneor more chemical moieties. For example, the partner nucleic acid strandmay be coupled to 1, 2, 3, 4, 5 or more chemical moieties. The couplingmay be covalent and may be direct or via a linker as described herein.In some embodiments, the second nucleic acid strand may be coupled orcouplable to a second chemical moiety. The second nucleic acid strandmay comprise a second coding sequence that encodes the second chemicalmoiety. In other embodiments, the second nucleic acid strand may not becoupled to a chemical moiety.

As described above, the chemical moieties that are coupled to the firstnucleic acid strand or the first and second nucleic acid strands form apharmacophore, when the strands self-assemble through hybridisation toform the library member.

The nucleic acid strand or the second nucleic acid strand may comprise aspacer region. The spacer region is non-hybridizable and may be called anon-hybridizable spacer.

The spacer region is an abasic region that does not hybridise tonucleotide sequences and is not a template for a nucleic acidpolymerase. Suitable spacer regions may comprise an abasicphosphodiester backbone or a linker, such as an alkyl chain,polyethylene glycol or other oligomer that spans the spacer region.

Suitable spacer regions may be obtained from commercial suppliers. Thespacer region may be located in a first nucleic acid strand at aposition that would otherwise hybridise with a coding sequence locatedin the second nucleic acid strand in the double stranded nucleic acidmolecule or may be located in the second nucleic acid strand at theregion that would otherwise hybridise with a coding sequence in thefirst nucleic acid strand. In some embodiments, regions complementary tothe first coding sequence; the first and second coding sequences; or thefirst, second and third coding sequences may be replaced by spacerregions.

A nucleic acid strand containing one or more spacer regions at positionscorresponding to coding sequences may hybridise to nucleic acid strandscontaining different coding sequences. This may be useful in theproduction of diversity in self-assembling libraries.

In some embodiments, the hybridisation of a first and a second nucleicacid strand may leave a single stranded overhanging region in the firstnucleic acid strand. The single-stranded region of the first nucleicacid strand may comprise a first coding sequence. The first nucleic acidstrand may further comprise a spacer region that corresponds in thedouble stranded nucleic acid molecule to a second coding region in thesecond nucleic acid strand, such that the first nucleic acid strand doesnot hybridize to the second coding region of the second nucleic acidstrand. A method may comprise;

-   -   extending the second nucleic acid strand along the first nucleic        acid strand,    -   such that the second nucleic acid strand incorporates the        complement of the first coding sequence.

The second nucleic acid strand of the library member may therebycomprise a first coding sequence encoding a first chemical moiety and asecond coding sequence encoding a second chemical moiety.

Suitable techniques for extending a nucleic acid strand along a templatenucleic acid strand are well known in the art. For example, the secondnucleic acid strand may be extended by addition of nucleotides forpolymerisation (normally in excess), preferably deoxynucleotides(dNTPs), and a polymerase (e.g. Taq or Klenow polymerase) in a suitablebuffer, incubated at a suitable temperature (e.g. 37° C. for Klenowpolymerase or 65° C. or 72° C. for Taq). In some embodiments, thenucleic acid strands of a nucleic acid encoded chemical sub-library maybe coupled to a single chemical moiety.

A method of producing a nucleic acid encoded chemical sub-library maycomprise,

-   -   (i) providing a first diverse population of chemical moieties,    -   (ii) coupling a nucleic acid strand to the diverse population of        chemical moieties,    -   (iii) contacting the nucleic acid strands coupled to the        chemical moieties in the population with identifier        oligonucleotides comprising coding sequences and one or more        adaptor oligonucleotides,    -   such that the adaptor oligonucleotides hybridize to the nucleic        acid strands and the identifier oligonucleotides to form        partially double-stranded complexes,    -   wherein each nucleic acid strand is contacted with an identifier        oligonucleotide that comprises a coding sequence that encodes        the chemical moiety coupled to the nucleic acid strand, and;    -   each adaptor oligonucleotide hybridizes to more than one nucleic        acid strand and more than one identifier oligonucleotide,    -   and    -   (iv) ligating the nucleic acid strands to the identifier        oligonucleotides in the partially double-stranded complexes,        such that the identifier oligonucleotides are incorporated into        the nucleic acid strands,    -   thereby producing a sub-library of different chemical moieties,        each chemical moiety being coupled to a nucleic acid strand        comprising a coding sequence that encodes the chemical moiety

In some preferred embodiments, all the nucleic acid strands arecontacted with the same adaptor oligonucleotide.

Following ligation, the adaptor may be removed, as described above. Insome preferred embodiments, the adaptor may be cleaved, for example bybase hydrolysis or enzymatic treatment.

The nucleic acid strand may comprise a spacer at a position thatcorresponds to a second coding sequence in a second nucleic acid strand.A method may further comprise;

-   -   (v) hybridizing the nucleic acid strands to second nucleic acid        strands to form a double-stranded complex,    -   wherein the second nucleic acid strands are coupled to a second        diverse population of chemical moieties, each second nucleic        acid strand comprising a second coding sequence that encodes the        chemical moiety that is coupled to it,    -   the position of the second coding sequence in the second nucleic        acid strands corresponding in the double-stranded complex to the        position of the spacer in the first nucleic acid strand in the        double-stranded complex, such that the second coding sequence        does not hybridise to the first nucleic acid strand,    -   (vi) extending the second nucleic acid strand along the nucleic        acid strand to produce a library comprising members having;    -   the first nucleic acid strand and the second nucleic acid strand        annealed together;    -   a chemical moiety from the first diverse population being        coupled to the first nucleic acid strand and a chemical moiety        from the second diverse population being coupled to the second        nucleic acid strand, said chemical moieties forming a        pharmacophore for screening,    -   wherein the second nucleic acid strand comprises a first coding        sequence that encodes the chemical moiety from the first diverse        population and a second coding sequence that encodes the        chemical moiety from the second diverse population.

Methods of the invention may be useful in the generation of nucleic acidencoded chemical libraries. Examples of suitable methods are shown inFIGS. 1A and 1B.

For example, a method of producing a nucleic acid encoded chemicallibrary may comprise,

-   -   (i) providing a first nucleic acid strand comprising a        non-hybridisable spacer and having a first chemical moiety        conjugated thereto,    -   (ii) contacting the first nucleic acid strand with an adaptor        and an identifier oligonucleotide comprising a coding sequence        encoding the first chemical moiety,    -   wherein the adaptor comprises a first annealing region portion        which hybridizes to the first nucleic acid strand, and a second        annealing region which hybridizes to the identifier        oligonucleotide to form a double-stranded complex comprising the        first nucleic acid strand, cleavable adaptor and identifier        oligonucleotide,    -   (iii) ligating the first nucleic acid strand to the identifier        oligonucleotide in the double-stranded complex, such that the        identifier oligonucleotide is incorporated into the first        nucleic acid strand; and    -   (iv) removing the adaptor to produce a first nucleic acid strand        linked to the chemical moiety and comprising a coding sequence        encoding the chemical moiety,    -   (v) repeating steps (i) to (iv) in series or in parallel using        different first chemical moieties and coding sequences and the        same adaptor to produce a diverse population of first chemical        moieties, each chemical moiety being coupled to a first nucleic        acid strand which comprises a first coding sequence encoding the        first chemical moiety,    -   (vi) contacting the diverse population of first chemical        moieties with a diverse population of second chemical moieties,        each second chemical moiety being coupled to a second nucleic        acid strand which comprises a second coding sequence encoding        the second chemical moiety coupled thereto, such that the first        and second nucleic acid strands hybridise to form a        double-stranded nucleic acid molecule,    -   wherein the position of the second coding sequence in the second        nucleic acid strands corresponds to the position of the        non-hybridisable spacer in the first nucleic acid strands, such        that the second coding sequence does not hybridise to the first        nucleic acid strands in the in the double-stranded nucleic acid        molecules, and    -   (vii) extending the second nucleic acid strands along the first        nucleic acid strands to produce a nucleic acid encoded chemical        library    -   each member of the library comprising;    -   a pharmacophore comprising a member of the diverse population of        first chemical moieties and a member of a diverse population of        second chemical moieties and;    -   a nucleic acid strand comprising a first coding sequence that        encodes the first chemical moiety of the pharmacophore and a        second coding sequence that encodes the second chemical moiety        of the pharmacophore.

In some embodiments, further chemical moieties may be coupled to one orboth of the nucleic acid strands, for example using a so-called “splitand pool” method. Examples of suitable methods are shown in FIGS. 2 and3. For example, steps (i) to (iv) may be repeated to couple a secondchemical moiety to the first nucleic acid strand and incorporate asecond coding sequence encoding the second chemical moiety into thefirst nucleic acid strand. For example, a method may further comprise;

-   -   (v) coupling a diverse population of second chemical moieties to        the first nucleic acid strands,    -   (vi) contacting the first nucleic acid strands coupled to the        second chemical moieties with a second adaptor and a second        identifier oligonucleotide comprising a coding sequence,    -   such that the second adaptor hybridizes to the first nucleic        acid strands and the identifier oligonucleotides to form        partially double-stranded complexes,    -   wherein each first nucleic acid strand is contacted with a        second identifier oligonucleotide that comprises a second coding        sequence that encodes the second chemical moiety that is coupled        to the first nucleic acid strand, and;    -   each second adaptor oligonucleotide hybridizes to more than one        first nucleic acid strand and more than one second identifier        oligonucleotide, and    -   (vii) ligating the first nucleic acid strands to the second        identifier oligonucleotides in the double-stranded complexes,        such that the second coding sequence identifier oligonucleotides        are incorporated into the nucleic acid strands.

The adaptor may then be removed, for example by cleavage and/orpurification, to produce a sub-library; each member of the sub-librarycomprising a first and a second chemical moiety coupled to a firstnucleic acid comprising coding sequences encoding the first and secondchemical moieties.

Step v) may be performed before step vi) or after step vii).

The second chemical moieties are coupled to the same ends of the nucleicacid strands as the first chemical moieties, so that the second chemicalmoieties and first chemical moieties form a pharmacophore on the samestrand for screening.

The second chemical moiety may be coupled to the first chemical moietyor to the first nucleic acid strand, either directly or through alinker.

Preferably, all the nucleic acid strands are contacted with a secondadaptor oligonucleotide having the same nucleotide sequence i.e. thesame second adaptor oligonucleotide sequence hybridises to all of thenucleic acid strands and second identifier oligonucleotides.

The second adaptor oligonucleotide may be the same as the first adaptoroligonucleotide or more preferably different.

The second adaptor hybridizes to the distal end of the nucleic acidstrand i.e. the end that is not linked to the chemical moiety. In someembodiments, the second adaptor may hybridise to a complementaryannealing sequence of the first identifier oligonucleotide which isincorporated into the nucleic acid strand. Steps (v) to (vii) may berepeated one or more times to couple further chemical moieties to thefirst nucleic acid strand and incorporate coding sequences encoding thefurther chemical moieties into the first nucleic acid strand, forexample as shown in FIGS. 3A and 3B. For example, a method may furthercomprise;

-   -   (viii) coupling a diverse population of further chemical        moieties to the first nucleic acid strands,    -   (ix) contacting the first nucleic acid strands coupled to the        further chemical moieties with a further adaptor oligonucleotide        and a further identifier oligonucleotide comprising a coding        sequence,    -   such that the further adaptor oligonucleotide hybridizes to the        nucleic acid strands and the identifier oligonucleotides to form        partially double-stranded complexes,    -   wherein each nucleic acid strand is contacted with a further        identifier oligonucleotide that comprises a further coding        sequence that encodes the further chemical moiety that is        coupled to the nucleic acid strand, and;    -   each further adaptor oligonucleotide hybridizes to more than one        nucleic acid strand and more than one further identifier        oligonucleotide, and    -   (x) ligating the nucleic acid strands to the further identifier        oligonucleotides in the double-stranded complexes, such that the        further coding sequence identifier oligonucleotides are        incorporated into the nucleic acid strands.

The adaptor may then be removed, for example by cleavage andpurification to produce a sub-library; each member of the sub-librarycomprising first, second and further chemical moieties coupled to anucleic acid strand comprising coding sequences encoding the first,second and further chemical moieties.

Step viii) may be performed before step ix) or after step x).

The further chemical moiety may be coupled to the same end of thenucleic acid strand as the first and second chemical moieties, so thatthe chemical moieties form a pharmacophore for screening. The furtherchemical moiety may be coupled to the first chemical moiety, the secondchemical moiety or the nucleic acid strand, either directly or through alinker.

Preferably, all the nucleic acid strands are contacted with a furtheradaptor oligonucleotide having the same nucleotide sequence i.e. thesame further adaptor oligonucleotide sequence hybridises to all of thenucleic acid strands and further identifier oligonucleotides.

The further adaptor may have the same sequence as the first and secondadaptors or more preferably, a different sequence.

The further adaptor hybridizes to the distal end of the nucleic acidstrand i.e. the end that is not linked to the chemical moiety. In someembodiments, the further adaptor may hybridise to a complementaryannealing sequence of the second identifier oligonucleotide which isincorporated into the nucleic acid strand.

Steps (viii) to (x) may be repeated one or more times to incorporate oneor more further coding sequences into the nucleic acid strand and couplethe nucleic acid strand to one or more chemical moieties. For example, anucleic strand may coupled to 3, 4, 5 or 6 or more chemical moieties.

A method may comprise repeating steps i) to iv), i) to vii) or i) to x)in series or in parallel using different first, second and/or furtherchemical moieties to produce a library comprising a diverse populationof library members having different combinations of the first, secondand further chemical moieties.

In some embodiments, n chemical moieties may be coupled to a nucleicacid strand as described above. The nth coding sequence (i.e. the codingsequence encoding the nth chemical moiety; the final chemical moiety tobe coupled to the strand) may be incorporated into the nucleic acidstrand by primer extension. A suitable method is shown in FIG. 3B.

For example, an identifier oligonucleotide comprising the nth codingsequence of a nucleic acid strand coupled to n chemical moieties (e.g.the second or further coding sequence) may be hybridised to the regionadjacent the 3′ end of the nucleic acid strand to produce asingle-stranded 5′ overhang of identifier oligonucleotide sequence thatcomprises the final coding sequence. The nucleic acid strand may then beextended 5′ to 3′ along the identifier oligonucleotide template toincorporate the complement of the final coding sequence into theextended nucleic acid strand. In some embodiments, the identifieroligonucleotide may be extended 5′ to 3′ along the nucleic strand toprovide a second nucleic strand hybridised to the nucleic strand.

Suitable methods of 5′ to 3′ extension of nucleic acid strands along atemplate, for example using DNA polymerases and active fragmentsthereof, are well known in the art.

A sub-library produced as described above may comprise a diversepopulation of library members comprising different chemical moieties ordifferent combinations of chemical moieties.

In some embodiments, a nucleic acid encoded chemical sub-library maydisplay pharmacophores consisting of two or more chemical moieties thatare coupled to a nucleic acid strand that includes coding sequencesencoding the two or more chemical moieties. A suitable method is shownin FIG. 3A. For example, a method of producing a member of a nucleicacid encoded chemical library may comprise;

-   -   (i) providing a first diverse population of chemical moieties,    -   (ii) coupling first nucleic acid strands to the diverse        population of chemical moieties,    -   (iii) contacting the first nucleic acid strands coupled to the        chemical moieties with an adaptor oligonucleotide and an        identifier oligonucleotide comprising a coding sequence,    -   such that the adaptor oligonucleotide hybridizes to the nucleic        acid strand and the identifier oligonucleotide to form a        partially double-stranded complex,    -   wherein each nucleic acid strand is contacted with an identifier        oligonucleotide that comprises a coding sequence that encodes        the chemical moiety coupled to the nucleic acid strand, and;    -   wherein all the nucleic acid strands are contacted with the same        adaptor oligonucleotide,    -   (iv) ligating the nucleic acid strands to the identifier        oligonucleotides in the partially double-stranded complexes,        such that the identifier oligonucleotides are incorporated into        the nucleic acid strands,    -   (v) coupling a diverse population of further chemical moieties        to the first nucleic acid strands,    -   (vi) contacting the first nucleic acid strands coupled to the        further chemical moieties with a further adaptor and a further        identifier oligonucleotide comprising a coding sequence,    -   such that the further adaptor hybridizes to the nucleic acid        strands and the identifier oligonucleotides to form partially        double-stranded complexes,    -   wherein each nucleic acid strand is contacted with a further        identifier oligonucleotide that comprises a further coding        sequence that encodes the further chemical moiety that is        coupled to the nucleic acid strand, and;    -   wherein all the first nucleic acid strands are contacted with        the same adaptor oligonucleotide, and    -   (vii) ligating the nucleic acid strands to the further        identifier oligonucleotides in the double-stranded complexes,        such that the further coding sequence identifier        oligonucleotides are incorporated into the nucleic acid strands.    -   (viii) optionally repeating steps (v) to (vii) one or more        times,    -   thereby producing a library member comprising first chemical        moiety and one or more further chemical moieties, said moieties        forming a pharmacophore for screening, and a nucleic acid strand        comprising a first coding sequence which encodes the first        chemical moiety and one or more further coding sequences which        encode the one or more further chemical moieties.

In some embodiments, the adaptor and the further adaptor or furtheradaptors may have the same nucleotide sequence. In preferredembodiments, the adaptor and the further adaptor or further adaptors mayhave different nucleotide sequences.

Preferably, steps (v) to (vii) are repeated once, so that thesub-library comprises members having three chemical moieties and anucleic acid strand comprising a first coding sequence which encodes thefirst chemical moiety, a second coding sequence which encodes the secondchemical moiety and a third coding sequence which encodes the thirdchemical moiety.

Preferably, the first nucleic acid strands of the sub-library arehybridised with a second nucleic acid strand to produce a nucleic acidencoded chemical library for screening that comprises double-strandednucleic acid.

The second nucleic acid strand may comprise spacers that correspond tothe first, second and/or further coding sequences in the first nucleicacid strand, so that the same second nucleic acid strand may behybridised to different first nucleic acid strands. In some embodiments,the second nucleic acid strand may be extended along the template of thefirst nucleic acid strand following hybridisation, such that itcomprises the complement of one or more of the first, second and/orfurther coding sequences. In some embodiments, the second nucleic acidstrand may not be coupled to chemical moieties.

In other embodiments, a sub-library of first nucleic acid strandscoupled to first and second diverse populations of chemical moieties,and optionally third or more diverse populations of chemical moieties,as described above, may hybridise or self-assemble to a sub-library ofsecond nucleic acid strands coupled to a further diverse population ofchemical moieties, and optionally additional diverse population ofchemical moieties, to produce a self-assembling library that displayspharmacophores formed by the chemical moieties that are coupled to boththe first and second nucleic acid strands. Suitable self-assemblinglibraries may be produced by the methods shown in FIGS. 2A to 2E. Forexample, a nucleic acid encoded chemical library may be produced by amethod comprising;

-   -   (i) providing a sub-library of first nucleic acid strands        coupled to first and second diverse populations of chemical        moieties (“first and second chemical moieties”),    -   wherein each first nucleic acid strand comprises a first coding        sequence which encodes the member of the first diverse        population of chemical moieties that is coupled to the first        nucleic acid strand,    -   (ii) contacting the first nucleic acid strands with an adaptor        oligonucleotide and first identifier oligonucleotides comprising        coding sequences, such that the adaptor oligonucleotide        hybridizes to the first nucleic acid strands and the first        identifier oligonucleotides to form partially double-stranded        complexes,    -   wherein each first nucleic acid strand is contacted with a first        identifier oligonucleotide comprising a coding sequence which        encodes the member of the second population of chemical moieties        that is coupled to the first nucleic acid strand, and;    -   wherein all the first nucleic acid strands in the sub-library        are contacted with the same adaptor oligonucleotide,    -   (iii) ligating the first nucleic acid strands to the first        identifier oligonucleotides in the complexes, such that the        second coding sequences are incorporated into the first nucleic        acid strands;    -   (iv) contacting the first nucleic acid strands with a nucleic        acid spacer strand, second identifier oligonucleotides, and a        sub-library of second nucleic acid strands coupled to a third        diverse population of chemical moieties (“third chemical        moieties”), thereby forming partially double-stranded complexes,    -   wherein each first nucleic acid strand is contacted with a        second identifier oligonucleotide comprising a third coding        sequence that encodes the member of the third population of        chemical moieties that is coupled to the second nucleic acid        strand, and;    -   wherein all the nucleic acid strands in the population are        contacted with the same nucleic acid spacer strand,    -   (v) ligating the first nucleic acid strand to the second        identifier oligonucleotide such that the third coding sequence        is incorporated into the nucleic acid strand; and,    -   (vi) optionally ligating the second nucleic acid strand to the        nucleic acid spacer strand,    -   thereby producing a library comprising pharmacophores labelled        with double-stranded nucleic acid molecules comprising first and        second nucleic acid strands.

In another example, a nucleic acid encoded chemical library may beproduced by a method comprising;

-   -   (i) providing a sub-library of first nucleic acid strands        coupled to first and second diverse populations of chemical        moieties (“first and second chemical moieties”),    -   wherein each first nucleic acid strand comprises a first coding        sequence which encodes the member of the first diverse        population of chemical moieties that is coupled to the first        nucleic acid strand,    -   (ii) contacting the first nucleic acid strands with an adaptor        oligonucleotide and first identifier oligonucleotides comprising        coding sequences, such that the adaptor oligonucleotide        hybridizes to the first nucleic acid strands and the first        identifier oligonucleotides to form partially double-stranded        complexes,    -   wherein each first nucleic acid strand is contacted with a first        identifier oligonucleotide comprising a coding sequence which        encodes the member of the second population of chemical moieties        that is coupled to the first nucleic acid strand, and;    -   wherein all the first nucleic acid strands in the sub-library        are contacted with the same adaptor oligonucleotide,    -   (iii) ligating the first nucleic acid strands to the first        identifier oligonucleotides in the complexes, such that the        second coding sequences are incorporated into the first nucleic        acid strands;    -   (iv) contacting the first nucleic acid strands with a        sub-library of second nucleic acid strands coupled to a third        diverse population of chemical moieties (“third chemical        moieties”), thereby forming partially double-stranded complexes,    -   wherein each second nucleic acid strand comprises spacer regions        at positions correspond to the first and second coding sequences        in the first nucleic acid strand and a third coding sequence        that encodes the member of the third population of chemical        moieties that is coupled to the second nucleic acid strand; said        third coding sequence forming a 5′ overhang in the partially        double-stranded complex,    -   (v) extending the first nucleic acid strand along the second        nucleic acid strand to incorporate the complement of the third        coding sequence into the first nucleic acid strand; and,    -   thereby producing a library comprising pharmacophores labelled        with double-stranded nucleic acid molecules comprising first and        second nucleic acid strands.

Each pharmacophore in the library is formed from the members of thefirst and second diverse populations of chemical moieties that arecoupled to the first nucleic acid strand of a library member and themember of the third diverse population of chemical moieties that iscoupled to the second nucleic acid strand of the library member.

The first nucleic acid strand of each library member comprises a firstcoding sequence that encodes the member of the first diverse populationof chemical moieties that is coupled to the first nucleic acid strand, asecond coding sequence encoding the member of the second diversepopulation of chemical moieties that is coupled to the first nucleicacid strand, and a third coding sequence encoding the member of thethird diverse population of chemical moieties that is coupled to thesecond nucleic acid strand of the library member.

A suitable second nucleic acid strand for use in a method describedabove hybridises to the first nucleic acid strand and may comprise;

-   -   a) a first hybridization portion which hybridizes to the first        nucleic acid strand,    -   b) a non-hybridizable spacer at a position that corresponds,        when the first and second strands are hybridised together, to        the position of the first coding sequence in the first nucleic        acid strand; and    -   c) a second hybridization portion which hybridizes to the first        nucleic acid strand.

In some preferred embodiments, identical second nucleic acid strands maybe coupled to all of the members of the third diverse population ofchemical moieties.

A suitable nucleic acid spacer strand for use in a method describedabove hybridises to the first nucleic acid strand and may comprise;

-   -   a) a first hybridization portion which hybridizes to the first        nucleic acid strand,    -   b) a non-hybridizable spacer at a position that corresponds,        when the first nucleic acid strand and the nucleic acid spacer        strand are hybridised together, to the position of the second        coding sequence in the first nucleic acid strand    -   c) a second hybridization portion which hybridizes to the first        nucleic acid strand; and,    -   d) a complementary annealing region which hybridizes to the        second identifier oligonucleotide.

In some preferred embodiments, the same nucleic acid spacer strand maybe used to produce all the members of the library i.e. all of themembers may be produced using identical nucleic acid spacer strands.

A suitable second identifier oligonucleotide for use in a methoddescribed above hybridises to the nucleic acid spacer strand and maycomprise;

-   -   a) a first annealing region which hybridizes to the nucleic acid        spacer strand,    -   b) a third coding sequence encoding the member of the third        diverse population of chemical moieties that is coupled to the        second nucleic acid strand.

In some preferred embodiments, the same first annealing region may beused in all of the second identifier oligonucleotides that are used toproduce the library i.e. all of the second identifier oligonucleotidesmay comprise an identical first annealing region. The diverse thirdcoding sequences will necessarily be employed in the second identifieroligonucleotides, depending on the identity of the member of the thirddiverse population of chemical moieties that is coupled to a particularsecond nucleic acid strand.

Following each ligation step, the adaptor oligonucleotide may be removedby cleavage and/or purification, as described above.

The first and second chemical moieties may be coupled to one of the 5′end or the 3′ end of the nucleic acid strand and the third chemicalmoiety may be coupled to the other of the 5′ end or the 3′ end of thepartner strand of the library member. In some preferred embodiments,first and second chemical moieties may be coupled to the 5′ end of thefirst nucleic acid strand and the third chemical moiety may be coupledto the 3′ end of the second nucleic acid strand.

Self-assembly of the sub-library of first nucleic acid strands and thesub-library of second nucleic acid strands by hybridisation as describedabove produces an encoded self-assembly chemical library comprising adiverse population of library members displaying pharmacophores formedfrom different combinations of the first, second and third chemicalmoieties coupled to the first and second nucleic acid strands as shownin FIG. 2A to 2E.

The sub-library of first nucleic acid strands may be produced by amethod comprising;

-   -   (i) providing a first nucleic acid strand having first and        second chemical moieties coupled thereto,    -   wherein the nucleic acid strand comprises a first coding        sequence which encodes the first chemical moiety,    -   (ii) contacting the nucleic acid strand with an adaptor        oligonucleotide and a first identifier oligonucleotide        comprising a second coding sequence that encodes the second        chemical moiety,    -   such that the adaptor oligonucleotide hybridizes to the nucleic        acid strand and the identifier oligonucleotide to form a        partially double-stranded complex,    -   (iii) ligating the nucleic acid strand to the first identifier        oligonucleotide in the complex, such that the second coding        sequence is incorporated into the nucleic acid strand; and,    -   (iv) repeating steps (i) to (iii) in series or in parallel to        using different first and second chemical moieties and first and        second coding sequences and the same adaptor oligonucleotide to        produce a diverse population of pairs of first and second        chemical moieties coupled to first nucleic acid strands,    -   each pair of chemical moieties being coupled to a first nucleic        acid strand which comprises a first coding sequence encoding the        first chemical moiety and a second coding sequence encoding the        second chemical moiety coupled thereto.

Examples of suitable methods are shown in FIGS. 2A, 2C and 2D.

The diverse population of pairs of first and second chemical moietiescoupled to first nucleic acid strands produced by step v) may then becombined or pooled into a single diverse population or sub-library.

In some embodiments, the sub-library of first nucleic acid strands maybe hybridised with a sub-library of second nucleic acid strands thatcomprise spacer regions and a third coding sequence.

The sub-library of second nucleic acid strands may be produced by amethod comprising;

-   -   (i) providing a second nucleic acid strand having a third        chemical moiety coupled thereto,    -   wherein the second nucleic acid strand comprises a first spacer        region at a position corresponding to the first coding sequence        in the first nucleic acid strand,    -   (ii) contacting the second nucleic acid strand with an adaptor        oligonucleotide and a nucleic acid spacer strand comprising a        second spacer region at a position corresponding to the second        coding sequence in the first nucleic acid strand,    -   such that the adaptor oligonucleotide hybridizes to the second        nucleic acid strand and the nucleic acid spacer strand to form a        partially double-stranded complex,    -   (iii) ligating the second nucleic acid strand to the nucleic        acid spacer strand in the complex, such that the second spacer        region is incorporated into the second nucleic acid strand;    -   (iv) contacting the second nucleic acid strand with an adaptor        oligonucleotide and a second identifier oligonucleotide        comprising a third coding sequence that encodes the third        chemical moiety,    -   such that the adaptor oligonucleotide hybridizes to the second        nucleic acid strand and the second identifier oligonucleotide to        form a partially double-stranded complex,    -   (v) ligating the second nucleic acid strand to the second        identifier oligonucleotide in the complex, such that the third        coding sequence is incorporated into the second nucleic acid        strand;    -   (vi) repeating steps (i) to (v) in series or in parallel using        different third chemical moieties and third coding sequences and        the same adaptor oligonucleotide to produce a diverse population        of third chemical moieties coupled to second nucleic acid        strands,    -   each third chemical moiety being coupled to a second nucleic        acid strand which comprises first and second spacer regions and        a third coding sequence encoding the third chemical moiety        coupled thereto.

An example of a suitable method is shown in FIG. 2D.

The third coding sequence (i.e. the coding sequence encoding the thirdchemical moiety) from the second nucleic acid of the members of thelibrary or its complement may be incorporated into the first nucleicacid strand of the members by primer extension. A method may comprise;

-   -   (vii) contacting the sub-library of first nucleic acid strand        strands to the sub-library of second nucleic acid strands,    -   the second nucleic acid strand hybridising to the first nucleic        acid strand to form a double-stranded complex having a 5′        overhang comprising the third coding sequence,    -   (viii) extending the first nucleic acid strand along the second        nucleic acid strand to incorporate the complement of the third        coding sequence into the first nucleic acid strand;    -   thereby producing library in which each member comprises a        pharmacophore formed by the first and second chemical moieties        coupled to the first nucleic acid strand and the third chemical        moiety coupled to the second nucleic acid strand; wherein the        first nucleic acid strand comprises first, second and third        coding sequences that encode the first, second and third        chemical moieties, respectively.

An example of a suitable method is shown in FIG. 2E.

In other embodiments, a separate identifier oligonucleotide comprisingthe third coding sequence may be employed. A method may comprise;

-   -   (vi) splitting the sub-library of first nucleic acid strands        into pools;    -   (vii) contacting a pool of the sub-library with a second nucleic        acid strand coupled to a third chemical moiety, a nucleic acid        spacer strand and a second identifier oligonucleotide comprising        a third coding sequence that encodes the third chemical moiety        that is coupled to the second nucleic acid strand,    -   the second nucleic acid strand and the nucleic acid spacer        strand hybridising to the first nucleic acid strand and the        second identifier oligonucleotide to form a partially        double-stranded complex,    -   wherein the second identifier oligonucleotide comprises a third        coding sequence that encodes the third chemical moiety that is        coupled to the second nucleic acid strand,    -   (viii) ligating the nucleic acid strands to the second        identifier oligonucleotide such that the third coding sequence        is incorporated into the first nucleic acid strand;    -   (ix) optionally ligating the second nucleic acid strand to the        nucleic acid spacer strand,    -   (x) repeating steps vi) to ix) in series or in parallel using        different third chemical moieties and third coding sequences,    -   wherein second nucleic acid strands and nucleic acid spacer        strands having the same nucleotide sequence are used for more        than one different third chemical moiety and second identifier        oligonucleotide, to produce multiple pools of the first nucleic        acid strands, the first nucleic strands in each pool being        hybridised to second nucleic acid strands coupled to different        third chemical moieties, and    -   (xi) combining the pools into a single diverse population or        library,    -   each member of the library comprising a pharmacophore formed by        the first and second chemical moieties coupled to the first        nucleic acid strand and the third chemical moiety coupled to the        second nucleic acid strand; wherein the first nucleic acid        strand comprises first, second and third coding sequences that        encode the first, second and third chemical moieties,        respectively.

An example of a suitable method is shown in FIG. 2A.

Preferably, identical second nucleic acid strands and nucleic acidspacer strands are used in all of the repetitions of step (x).

In some embodiments, the first, second or further adaptoroligonucleotide may be removed following each ligation step. Forexample, the adaptor oligonucleotide may be removed by purificationand/or fragmentation, as described above.

The third coding sequence (i.e. the coding sequence encoding the thirdchemical moiety) may be incorporated into the first nucleic acid strandby primer extension, for example as set out in FIG. 2C. A method maycomprise;

-   -   (vi) splitting the sub-library of first nucleic acid strands        into pools;    -   (vii) contacting a pool of the sub-library with a second nucleic        acid strand coupled to a third chemical moiety, a nucleic spacer        strand and a second identifier oligonucleotide comprising a        third coding sequence that encodes the third chemical moiety        that is coupled to the second nucleic acid strand,    -   the second nucleic acid strand, nucleic spacer strand and second        identifier oligonucleotide hybridising to the first nucleic acid        strand to form a double-stranded complex having a 5′ overhang        comprising the third coding sequence,    -   (viii) extending the first nucleic acid strand along the second        identifier oligonucleotide to incorporate the complement of the        third coding sequence into the first nucleic acid strand;    -   (ix) optionally ligating the second nucleic acid strand to the        nucleic acid spacer strand and second identifier        oligonucleotide,    -   (x) repeating steps vi) to ix) in series or in parallel using        different third chemical moieties and third coding sequences,        wherein second nucleic acid strands and nucleic acid spacer        strands having the same nucleotide sequence are used for more        than one different third chemical moiety and second identifier        oligonucleotide, to produce multiple pools of first nucleic acid        strands, the first nucleic strands in each pool comprising        different third coding sequences and being hybridised to second        nucleic acid strands coupled to different third chemical        moieties, and    -   (xi) combining the pools into a single diverse population or        library,    -   each member of the library comprising a pharmacophore formed by        the first and second chemical moieties coupled to the first        nucleic acid strand and the third chemical moiety coupled to the        second nucleic acid strand; wherein the first nucleic acid        strand comprises first, second and third coding sequences that        encode the first, second and third chemical moieties,        respectively.

In other embodiments, the adaptor oligonucleotide forms part of anucleic acid spacer strand that remains hybridised to the first nucleicacid strand and is optionally ligated to the second nucleic acid strand.A nucleic acid encoded chemical library may be produced by a methodcomprising;

-   -   (i) providing a sub-library of first nucleic acid strands        coupled to first and second diverse populations of chemical        moieties (“first and second chemical moieties”),    -   wherein each first nucleic acid strand comprises a first coding        sequence which encodes the member of the first diverse        population of chemical moieties that is coupled to the first        nucleic acid strand,    -   (ii) contacting the first nucleic acid strands with first        identifier oligonucleotides comprising second coding sequences        and one or more nucleic acid spacer strands, such that the first        nucleic acid spacer strands hybridize to the first nucleic acid        strands and the first identifier oligonucleotides to form        partially double-stranded complexes,    -   wherein each first nucleic acid strand is contacted with a first        identifier oligonucleotide comprising a second coding sequence        which encodes the member of the second population of chemical        moieties that is coupled to the first nucleic acid strand, and;    -   each nucleic acid spacer strand hybridises to more than one        different first identifier oligonucleotide and first nucleic        acid strands coupled to more than one different second chemical        moiety,    -   (iii) ligating the first nucleic acid strands to the first        identifier oligonucleotides in the complexes, such that the        second coding sequences are incorporated into the first nucleic        acid strands;    -   (v) contacting the first nucleic acid strands hybridised to the        nucleic acid spacer strand with a sub-library of second nucleic        acid strands coupled to a third diverse population of chemical        moieties (“third chemical moieties”) and second identifier        oligonucleotides, thereby forming double-stranded complexes,    -   wherein each first nucleic acid strand is contacted with a        second identifier oligonucleotide that comprises a third coding        sequence that encodes the member of the third population of        chemical moieties that is coupled to the second nucleic acid        strand that is contacted therewith, and;    -   (vi) ligating the first nucleic acid strand to the second        identifier oligonucleotide such that the third coding sequence        is incorporated into the first nucleic acid strand; and,    -   (vii) optionally ligating the second nucleic acid strand to the        nucleic acid spacer strand,    -   thereby producing a library comprising pharmacophores formed by        the first and second chemical moieties coupled to the first        nucleic acid strand and the third chemical moiety coupled to the        second nucleic acid strand; wherein the first nucleic acid        strand comprises first, second and third coding sequences that        encode the first, second and third chemical moieties,        respectively.

An example of a suitable method is shown in FIG. 2B.

Preferably, all the first nucleic acid strands in the sub-library arecontacted with an identical nucleic acid spacer strand.

Suitable second nucleic acid strands are described above.

Preferably, identical second nucleic acid strands are coupled to all ofthe members of the third diverse population of chemical moieties.

A suitable nucleic acid spacer strand hybridises to the first nucleicacid strand and may comprise;

-   -   a) a first hybridization portion which hybridizes to the first        nucleic acid strand,    -   b) a first non-hybridizable spacer at a position that        corresponds, when the first nucleic acid strand and the nucleic        acid spacer strand are hybridised together, to the position of        the second coding sequence in the first nucleic acid strand    -   c) a second hybridization portion which hybridizes to the first        nucleic acid strand;    -   d) a first annealing region which hybridizes to the second        identifier oligonucleotide,    -   e) a second non-hybridizable spacer at a position that        corresponds, when the second identifier oligonucleotide and the        nucleic acid spacer strand are hybridised together, to the        position of the third coding sequence in the second identifier        oligonucleotide and;    -   f) a second annealing region which hybridizes to the second        identifier oligonucleotide.

In some preferred embodiments, the same nucleic acid spacer strandsequence may be used to produce all the members of the library i.e. allof the members may be produced using identical nucleic acid spacerstrands.

A suitable second identifier oligonucleotide for use in a methoddescribed above hybridises to the nucleic acid spacer strand and maycomprise;

-   -   a) a first complementary annealing region which hybridizes to        the nucleic acid spacer strand,    -   b) a third coding sequence encoding the member of the third        diverse population of chemical moieties that is coupled to the        second nucleic acid strand and;    -   a) a second complementary annealing region which hybridizes to        the nucleic acid spacer strand.

In some preferred embodiments, all of the second identifieroligonucleotides that are used to produce the library may comprise thesame first and second complementary annealing regions i.e. all of thesecond identifier oligonucleotides may comprise an identical first andsecond annealing regions. This allows identical nucleic acid spacerstrands to be used for all second identifier oligonucleotides. Diversethird coding sequences will necessarily be employed in the secondidentifier oligonucleotides, depending on the identity of the member ofthe third diverse population of chemical moieties that is coupled to aparticular second nucleic acid strand.

As described above, in some embodiments, a sub-library of second nucleicacid strands comprising one or more nucleic acid spacer strands andsecond identifier oligonucleotide sequences may be produced beforehybridisation to the sub-library of first nucleic acid strands. In otherembodiments, one or more nucleic acid spacer strands and secondidentifier oligonucleotide sequences may be ligated to the secondnucleic acid strands after hybridisation to the first nucleic acidstrands.

As described above, self-assembly of the sub-library of first nucleicacid strands and the sub-library of second nucleic acid strands byhybridisation as described above produces an encoded self-assemblychemical library comprising a diverse population of library membersdisplaying pharmacophores formed from different combinations of thefirst, second and third chemical moieties coupled to the first andsecond nucleic acid strands.

The sub-library of first nucleic acid strands may be produced by amethod comprising;

-   -   (i) providing a first nucleic acid strand having first and        second chemical moieties coupled thereto,    -   wherein the nucleic acid strand comprises a first coding        sequence which encodes the first chemical moiety,    -   (ii) contacting the nucleic acid strand with a nucleic acid        spacer strand and a first identifier oligonucleotide comprising        a second coding sequence that encodes the second chemical        moiety,    -   such that the nucleic acid spacer strand hybridizes to the        nucleic acid strand and the first identifier oligonucleotide to        form a partially double-stranded complex,    -   (iii) ligating the nucleic acid strand to the first identifier        oligonucleotide in the complex, such that the second coding        sequence is incorporated into the nucleic acid strand in the        complex; and,    -   (iv) repeating steps (i) to (iii) in series or in parallel using        different first and second chemical moieties and first and        second coding sequences, wherein each nucleic acid spacer strand        hybridises to first nucleic acid strands coupled to more than        one combination of first and second chemical moieties and first        identifier oligonucleotides comprising more than one different        second coding sequence, to produce a diverse population of pairs        of first and second chemical moieties coupled to first nucleic        acid strands,    -   each pair of chemical moieties being coupled to a first nucleic        acid strand which comprises a first coding sequence encoding the        first chemical moiety and a second coding sequence encoding the        second chemical moiety coupled thereto, the nucleic acid spacer        strand being hybridised to the first nucleic acid strand.

Suitable methods are illustrated in FIG. 2B.

Preferably, an identical nucleic acid spacer strand is used in eachrepetition of step (iv).

In some embodiments, the nucleic acid spacer strand remains hybridisedto the first nucleic acid strand.

In other embodiments, the nucleic acid spacer strand may be removed, forexample, by denaturation and purification, and then rehybridised to thefirst nucleic acid strand.

The diverse population of pairs of first and second chemical moietiescoupled to first nucleic acid strand/nucleic acid spacer strandcomplexes, as produced by step (iv), may then be combined or pooled intoa single sub-library.

A method may comprise;

-   -   (vi) splitting the sub-library of first nucleic acid strands and        nucleic acid spacer strand complexes into pools;    -   (vii) contacting a pool of the sub-library with a second nucleic        acid strand coupled to a third chemical moiety and a second        identifier oligonucleotide comprising a third coding sequence        that encodes the third chemical moiety, thereby forming a        double-stranded complex comprising the first and second nucleic        acid strands, the nucleic acid spacer strand, the second        identifier oligonucleotide and the first, second and third        chemical moieties,    -   wherein the second identifier oligonucleotide comprises a third        coding sequence that encodes the third chemical moiety that is        coupled to the second nucleic acid strand,    -   (viii) ligating the first nucleic acid strands to the second        identifier oligonucleotide such that the third coding sequence        is incorporated into the first nucleic acid strand;    -   (ix) optionally ligating the second nucleic acid strand to the        nucleic acid spacer strand,    -   (x) repeating steps vi) to ix) in series or in parallel using        different third chemical moieties and third coding sequences,        and identical second nucleic acid strands and nucleic acid        spacer strands, thereby producing a library of diverse        pharmacophores,    -   wherein each nucleic acid spacer strand hybridises to second        nucleic acid strands that are coupled to more than one different        third chemical moiety and second identifier oligonucleotides        that comprise more than one different third coding sequence,    -   each member of the library comprising a pharmacophore formed by        the first and second chemical moieties coupled to the first        nucleic acid strand and the third chemical moiety coupled to the        second nucleic acid strand; wherein the first nucleic acid        strand comprises first, second and third coding sequences that        encode the first, second and third chemical moieties,        respectively.

Preferably, nucleic acid spacer strands having identical nucleotidesequences are used for each repetition of step (x).

Suitable methods are illustrated in FIG. 2B.

Libraries produced by the methods described above may comprise 500 ormore, 1000 or more, 10000 or more, 100000 or more or 1000000 or moredifferent library members, each different member displaying a differentpharmacophore formed from a different combination of chemical moieties.

Once the encoded chemical library members have been synthesised asdescribed above, they can be combined into an encoded chemical library,for example by including the library members together in a single vesselor single reaction mixture. This facilitates screening of the chemicallibrary.

Another aspect of the invention provides a method of generating anucleic acid encoded chemical library comprising;

-   -   producing multiple diverse library members using a method        described above and    -   combining the library members to produce a chemical library.

Within a chemical library, members may include nucleic acid strandswhich are coupled to the same number and type of chemical moiety butwhich are linked in a different order to each nucleic acid strand. Forexample, where a nucleic acid strand is coupled to two chemicalmoieties, A and B, some nucleic acid strands may include the moietieslinked in the order A-B, where A is distal to the nucleic acid strandand B is proximal to the nucleic acid strand, while others may containthe same two chemical moieties linked in the order B-A where B is distalto the nucleic acid strand and A is proximal to the nucleic acid strand.Assembly of each of these strands individually with a partner strandcoupled to a single moiety ‘C’ will produce two library members havingpharmacophores with different structures, even though they are composedof the same chemical moieties.

The same principle applies to chemical library members which includethree chemical moieties, A′, B′ and C′, where members may include themoieties linked as A′-B′-C′, A′-C′-B′, B′-A′-C′, B′-C′-A′, C′-A′-B′and/or C′-B′-A′ (ordered as proximal-middle-distal with respect to thenucleic acid strand in each case). Other arrangements of chemicalmoieties are possible, for example A′ and B′ may both be linked to C′but not to each other, or all of A′, B′ and C′ may form a covalentlylinked compound.

The same principle applies to chemical library members having four, fiveor more chemical moieties. Thus, it can be seen that the number ofcombinations of chemical moieties in the pharmacophore is increasedwhich can aid selection.

The number of different members in a chemical library represents thecomplexity of a library and is defined by number of different chemicalmoieties, the number of chemical moieties in each pharmacophore, andtherefore the number of different pharmacophores in the library. Thenumber of different pharmacophores of any particular library can bedetermined by multiplying the number of different types of chemicalmoieties together. For example, if each library member has two chemicalmoieties in the pharmacophore, and there are twenty types of eachchemical moiety, then the resulting library has 400 members. If, forexample, there are three chemical moieties in the pharmacophore, each ofwhich has twenty variants, then the resulting library has 8000 members.

The relative amounts of the individual chemical moieties within thelibrary can vary from about 0.2 equivalents to about 10 equivalents,where an equivalent represents the average amount of a chemical moietywithin the library. Preferably each chemical moiety is present in thelibrary in approximately equimolar amounts.

If desired, the members of a chemical library may be linked to a solidsupport such as a bead, array or other substrate surface. Alternativelythe library members can be free in solution.

One exemplary use for a chemical library is for lead optimization. Leadoptimization may involve combining a known pharmacophore, formed fromone or more chemical moieties with one or more further chemicalmoieties, as described herein with the aim of improving thecharacteristics of the known pharmacophore, for example the bindingaffinity. In this case, nucleic acid strands from a first and secondsub-library may be hybridized to form a library. The first sublibrarymay comprise library members which are coupled to the knownpharmacophore and the second sublibrary comprises members coupled to oneor more candidate chemical moieties. The second sublibrary generallycomprises a variety of different chemical moieties, because thisincreases the variety of structure in the pharmacophores of theassembled library members. The identities of the chemical moieties inthe resultant pharmacophore are encoded into the library member usingthe methods described herein.

An encoded chemical library generated according to the methods of thepresent invention provides a repertoire of chemical diversity in whicheach chemical moiety is linked to a identifier oligonucleotide thatfacilitates identification of the chemical moiety. The library may beused to screen for pharmacophores with particular properties, e.g.pharmacophores that bind a target molecule e.g. a protein. By screeningan encoded chemical library, it is possible to identify optimisedchemical structures that participate in binding interactions with abiological macromolecule by drawing upon a repertoire of structuresrandomly formed by the association of diverse chemical moieties withoutthe necessity of either synthesising them one at a time or knowing theirinteractions in advance.

Encoded chemical libraries produced as described herein may be used in avariety of such methods. For example, the library can be used in amethod for identifying a pharmacophore that participates in apreselected binding interaction with a biological macromolecule.

A method for identifying a pharmacophore which binds to a target ofinterest comprises the following steps:

-   -   (a) admixing a chemical library produced as described above with        a preselected biological macromolecule under binding conditions        (i.e., a binding reaction admixture) for a time period        sufficient for the biological macromolecule to interact with the        library and form a binding reaction complex with at least one        member thereof;    -   (b) isolating the binding reaction complex from the library        admixture to form an isolated complex;    -   (c) determining the coding sequences of the nucleic acid        moieties present in the isolated binding reaction complex,    -   thus identifying the chemical moieties that participated in the        binding reaction.

A typical biological macromolecule exhibiting a preselected bindinginteraction can be any of a variety of molecules (e.g. proteins) thatbind selectively to another molecule, including antibodies to antigens,lectins to oligosaccharides, receptors to ligands, enzymes to substratesand the like mediators of molecular interactions. Therefore, apreselected binding interaction is defined by the selection of thebiological macromolecule with which a library member is to bind.

The assembly of double-stranded libraries displaying pharmacophoresformed from chemical moieties on each of the strands allows theproduction of libraries containing large numbers of differentpharmacophores. For example, a method for producing a nucleic acidencoded chemical library may comprise,

-   -   (i) producing a first diverse population (A) of one or more        chemical moieties coupled to first nucleic acid strands using a        method described above,    -   (ii) producing a second diverse population (B) of one or more        chemical moieties coupled to second nucleic acid strands using a        method described above,    -   wherein the first nucleic acid strands hybridise to the second        nucleic acid strands to form library members, such that the        chemical moieties coupled to the first and second nucleic acid        strands of each library member form a pharmacophore,    -   (iii) combining the first and second diverse populations to        produce a library of library members comprising a        double-stranded nucleic acid molecule (A×B).

As described above, the first and second nucleic acid strands maycomprise one or more regions which are complementary to each otherallowing self-assembly when the diverse populations are combined.

Another aspect of the invention provides a nucleic acid encoded chemicallibrary comprising library members produced by a method described above.

Another aspect of the invention provides a method of screening a nucleicacid encoded chemical library comprising;

-   -   producing a nucleic acid encoded chemical library using a method        described above,    -   contacting the library with a target molecule and    -   selecting one or more library members which bind to the target.

The target molecule is a molecule which the pharmacophore is a candidatefor interacting with. The target molecule may be a biological moleculeas described herein or any other molecule of interest.

The library is contacted with a target molecule under binding conditionsfor a time period sufficient for the target molecule to interact withthe library and form a binding reaction complex with a least one memberthereof.

Binding conditions are those conditions compatible with the knownnatural binding function of the target molecule. Those compatibleconditions are buffer, pH and temperature conditions that maintain thebiological activity of the target molecule, thereby maintaining theability of the molecule to participate in its preselected bindinginteraction. Typically, those conditions include an aqueous, physiologicsolution of pH and ionic strength normally associated with the targetmolecule of interest.

For example, where the binding interaction is to identify a member inthe library able to bind an antibody molecule, the preferred bindingconditions would be conditions suitable for the antibody to immunoreactwith its immunogen, or a known immunoreacting antigen. For a receptormolecule, the binding conditions would be those compatible withmeasuring receptor ligand interactions.

A time period sufficient for the admixture to form a binding reactioncomplex is typically that length of time required for the biologicalmacromolecule to interact with its normal binding partner underconditions compatible with interaction. Although the time periods canvary depending on the molecule and its respective concentration,admixing times are typically for at least a few minutes, and usually notlonger than several hours, although nothing is to preclude using longeradmixing times for a binding reaction complex to form.

A binding reaction complex is a stable product of the interactionbetween a target molecule and a pharmacophore as described herein. Theproduct is referred to as a stable product in that the interaction ismaintained over sufficient time that the complex can be isolated fromthe rest of the members of the library without the complex becomingsignificantly disassociated.

The admixture of a library of the invention with a target molecule canbe in the form of a heterogeneous or homogeneous admixture. Thus, themembers of the library can be in the solid phase with the targetmolecule present in the liquid phase. Alternatively, the target moleculecan be in the solid phase with the members of the library present in theliquid phase. Still further, both the library members and the targetmolecule can be in the liquid phase.

The selected library members may be isolated and/or purified.

A binding reaction complex may be isolated from the binding reactionadmixture by any separation means that is selective for the complex,thereby isolating that library member, or members, which has/have boundto the target. There are a variety of separation means, depending on thestatus of the target.

For example, a target which is a biological macromolecule may beprovided in admixture in the form of a solid phase reagent, i.e.,affixed to a solid support, and thus can readily be separated from theliquid phase, thereby removing the majority of library members.Separation of the solid phase from the binding reaction admixture canoptionally be accompanied by washes of the solid support to rinselibrary members having lower binding affinities off the solid support.

Alternatively, for a homogeneous liquid binding reaction admixture, asecondary binding means specific for the biological macromolecule can beutilized to bind the molecule and provide for its separation from thebinding reaction admixture.

For example, an immobilised antibody immunospecific for the biologicalmacromolecule can be provided as a solid phase-affixed antibody to thebinding reaction admixture after the binding reaction complex is formed.The immobilised antibody immunoreacts with the biological macromoleculepresent in the binding reaction admixture to form an antibody-biologicalmacromolecule immunoreaction complex. Thereafter, by separation of thesolid phase from the binding reaction admixture, the immunoreactioncomplex, and therefore any binding reaction complex, is separated fromthe admixture to form isolated library member.

Alternatively, a binding means can be operatively linked to targetmolecule to facilitate its retrieval from the binding reactionadmixture. Exemplary binding means are one of the following highaffinity pairs: biotin-avidin, protein A-Fc receptor, ferritin-magneticbeads, and the like. Thus, the target is operatively linked (conjugated)to biotin, protein A, ferritin and the like binding means, and thebinding reaction complex is isolated by the use of the correspondingbinding partner in the solid phase, e.g., solid-phase avidin,solid-phase Fc receptor, solid phase magnetic beads and the like.

The use of solid supports on which to operatively link proteinaceousmolecules is generally well known in the art. Useful solid supportmatrices are well known in the art and include cross-linked dextran suchas that available under the tradename SEPHADEX from Pharmacia FineChemicals (Piscataway, N.J.); agarose, borosilicate, polystyrene orlatex beads about 1 micron to about 5 millimeters in diameter, polyvinylchloride, polystyrene, cross-linked polyacrylamide, nitrocellulose ornylon-based webs such as sheets, strips, paddles, plates microtiterplate wells and the like insoluble matrices.

The nucleic acid strand or the partner nucleic acid strand of theselected library members may be sequenced to identify the chemicalmoieties that form the pharmacophore displayed by the selected librarymembers.

The identifier oligonucleotides of the library members that bind to thetarget molecules may be amplified by PCR, which allows very low amountsof template nucleic acid to be detected. Subsequent decoding of theenriched nucleic acid uses either nucleic acid sequencing orhybridisation to oligonucleotide microarrays, depending on thearchitecture of the library and its size.

A preferred method for decoding is the use of high throughput sequencingmethods, such as the 454-Roche Genome Sequencer system. For sequencingwith the 454-Roche Genome Sequencer system, PCR products have to containsuitable adaptor sequences at their extremities (called adaptor sequenceA and B), which can be either added after a PCR reaction by ligation, orthey can be incorporated in the PCR reactions, if the PCR primerscontain on their 5′-ends sequences corresponding to an adaptor region.The next step of a particular sequencing process is the annealing of PCRamplicons on nucleic acid Capture Beads, emulsification of beads and PCRreagents in water-in-oil microreactors, and clonal emPCR amplificationinside these microreactors. After breaking of the emulsion, the Capturebeads are mixed with Enzyme Beads, and loaded on a PicoTiterPlate.Pyrosequencing allows the recording of individual sequences for eachnucleic acid species displayed at Capture Beads, trapped in the wells ofPicoTiterPlates. This allows the parallel sequencing of a vast amount(typically more than 100,000 per PicoTiterPlate) of individual nucleicacid species at a time. With further improvement of the sequencingtechnology, it will be possible to sequence more than 1,000,000individual nucleic acid species at a time.

Further details and examples of the use of library screening techniqueshave been described in the art [1] to [9].

Suitable primers may be primers which bind to primer regions in thenucleic acid strand or partner strand.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.

Other aspects and embodiments of the invention provide the aspects andembodiments described above with the term “comprising” replaced by theterm “consisting of” and the aspects and embodiments described abovewith the term “comprising” replaced by the term “consisting essentiallyof”.

It is to be understood that the application discloses all combinationsof any of the above aspects and embodiments described above with eachother, unless the context demands otherwise. Similarly, the applicationdiscloses all combinations of the preferred and/or optional featureseither singly or together with any of the other aspects, unless thecontext demands otherwise.

Modifications of the above embodiments, further embodiments andmodifications thereof will be apparent to the skilled person on readingthis disclosure, and as such these are within the scope of the presentinvention.

All documents and sequence database entries mentioned in thisspecification are incorporated herein by reference in their entirety forall purposes.

“and/or” where used herein is to be taken as specific disclosure of eachof the two specified features or components with or without the other.For example “A and/or B” is to be taken as specific disclosure of eachof (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

Certain aspects and embodiments of the invention will now be illustratedby way of example and with reference to the figures described below.

FIG. 1A shows an encoding strategy for a chemical library member using achimeric cleavable adaptor as described herein. In this scheme, anucleic acid strand is first coupled to a chemical moiety (or ‘buildingblock’). The nucleic acid strand contains a non-coding spacer region. Acleavable chimeric adaptor is used to couple an identifieroligonucleotide to the distal end of the nucleic acid strand withrespect to the moiety. The identifier oligonucleotide contains a codingsequence (codeB) which encodes the identity of the chemical moietyattached to the nucleic acid strand. The adaptor hybridizes tocomplementary bases on the distal end of the nucleic acid strand andproximal end of the identifier oligonucleotide. This brings an end ofthe identifier oligonucleotide into proximity to an end of the nucleicacid strand, such that ligation can occur under suitable conditions. Theends of the identifier oligonucleotide and nucleic acid strand are thenligated and the adaptor is removed. A partner nucleic acid strand isthen hybridized to the first nucleic acid strand. The partner nucleicacid strand is coupled to a further chemical moiety and includes anidentifier oligonucleotide containing a coding sequence (codeA) encodingthe identity of its chemical moiety. The nucleotide sequence of thepartner stand is then extended by polymerase-mediated fill-in so thatthe coding sequence encoding the identity of the first chemical moietyis located on the same strand as the coding sequence for the secondchemical moiety, in this case the partner strand.

The encoded chemical library member, which contains a pharmacophorecomprising two chemical moieties, can then be used for selectionexperiments on a target of interest. Following selection, candidatechemical library members are decoded by PCR amplification of the partnernucleic acid strand, which contains the coding sequences of bothchemical moieties. The spacer region located in the nucleic acid strandprevents amplification of this strand.

FIG. 1B shows an alternative strategy to that described in FIG. 2A.Here, the adaptor is used to couple an identifier oligonucleotidecontaining a coding sequence to the nucleic acid strand before thechemical moiety encoded by the coding sequence is coupled to the nucleicacid strand. Construction of the encoded library member then proceeds asin FIG. 1A.

FIG. 2A shows an encoding strategy for the production of a threebuilding block pharmacophore library in which the building blocks arecoupled to both strands of the members. A nucleic acid strand is firstcoupled to first and second chemical moieties (‘building blocks’). Thenucleic acid strand contains coding sequence (codeA) encoding the firstchemical moiety. A cleavable chimeric adaptor is used to couple anidentifier oligonucleotide to the distal end of the nucleic acid strandwith respect to the moieties. The identifier oligonucleotide contains acoding sequence (codeB) which encodes the identity of the secondchemical moiety attached to the nucleic acid strand. The adaptorhybridizes to complementary bases on the distal end of the nucleic acidstrand and proximal end of the identifier oligonucleotide forming acomplex between identifier oligonucleotide, nucleic acid strand andadaptor. This brings an end of the identifier oligonucleotide intoproximity to an end of the nucleic acid strand, such that ligation canoccur under suitable conditions. The ends of the identifieroligonucleotide and nucleic acid strand are then ligated and the adaptoris removed. The nucleic acid strand is then contacted with a partnernucleic acid strand, a nucleic acid spacer strand and a secondidentifier oligonucleotide to form a complex. The partner nucleic acidstrand is coupled to a third chemical moiety. The partner nucleic acidstrand hybridizes to the nucleic acid strand thought complementaryregions and includes a spacer region which does not hybridize to acoding region (codeA) of the nucleic acid strand. The nucleic acidspacer strand also hybridises to the nucleic acid strand and includes aspacer region which does not hybridize to the further coding region(codeB) of the nucleic acid strand. The second identifieroligonucleotide contains a coding sequence (codeC) encoding the identityof the third chemical moiety. The second identifier oligonucleotidehybridises to complementary regions on the nucleic acid spacer tag. Thesecond identifier oligonucleotide is then ligated to the nucleic acidstrand and the nucleic acid spacer strand is ligated to the partnernucleic acid strand. Coding information for all three chemical moietiesin the pharmacophore is now encoded on the nucleic acid strand.

FIG. 2B shows an alternative strategy for the production of a threebuilding block pharmacophore library in which the building blocks arecoupled to both strands of the members.

Here, a nucleic acid strand is first coupled to first and secondchemical moieties (‘building blocks’). The nucleic acid strand containsa first coding sequence (codeA) encoding a first chemical moiety. Thenucleic acid strand is then contacted with a nucleic acid spacer strandand a first identifier oligonucleotide to form a complex. The firstidentifier oligonucleotide contains a second coding sequence encodingthe identity of the second chemical moiety. The nucleic acid spacerstrand contains first and second non-hybridizable spacer regions at aposition in the nucleic acid spacer strand corresponding to the positionof the second coding sequence in and a further, third coding sequence inthe nucleic acid strand. The nucleic acid spacer strand hybridizes tothe nucleic acid strand and first identifier oligonucleotide throughcomplementary regions. The nucleic acid strand is then ligated to thefirst identifier oligonucleotide. The complex comprising the nucleicacid spacer strand and nucleic acid strand is then contacted with asecond identifier oligonucleotide and partner nucleic acid strand, whichhybridize through complementary regions to form a complex. In thecomplex each coding region is located in a position corresponding to anon-hybridizable spacer region. The partner nucleic acid strand iscoupled to a third chemical moiety and contains a spacer region at aposition in the nucleic acid partner strand corresponding to theposition of the first coding sequence in the nucleic acid strand. Thesecond identifier oligonucleotide contains a third coding sequenceencoding the identity of the third chemical moiety.

The partner nucleic acid strand may be ligated to the nucleic acidspacer strand and the second identifier oligonucleotide is ligated tothe nucleic acid strand.

FIG. 2C shows an alternative strategy for the production of a threebuilding block pharmacophore library in which the building blocks arecoupled to both strands of the members.

A nucleic acid strand is first coupled to first and second chemicalmoieties (‘building blocks’). The nucleic acid strand contains codingsequence (codeA) encoding the first chemical moiety. A cleavablechimeric adaptor is used to couple an identifier oligonucleotide to thedistal end of the nucleic acid strand with respect to the moieties. Theidentifier oligonucleotide contains a coding sequence (codeB) whichencodes the identity of the second chemical moiety attached to thenucleic acid strand. The adaptor hybridizes to complementary bases onthe distal end of the nucleic acid strand and proximal end of theidentifier oligonucleotide forming a complex between identifieroligonucleotide, nucleic acid strand and adaptor. This brings an end ofthe identifier oligonucleotide into proximity to an end of the nucleicacid strand, such that ligation can occur under suitable conditions. Theends of the identifier oligonucleotide and nucleic acid strand are thenligated and the adaptor is removed. The nucleic acid strand is thencontacted with a partner nucleic acid strand, a nucleic acid spacerstrand and a second identifier oligonucleotide to form a complex. Thepartner nucleic acid strand is coupled to a third chemical moiety. Thepartner nucleic acid strand hybridizes to the nucleic acid strandthrough complementary regions and includes a spacer region which doesnot hybridize to a coding region (codeA) of the nucleic acid strand. Thenucleic acid spacer strand also hybridises to the nucleic acid strandand includes a spacer region which does not hybridize to the furthercoding region (codeB) of the nucleic acid strand. The second identifieroligonucleotide contains a coding sequence (codeC) encoding the identityof the third chemical moiety. The proximal end of the second identifieroligonucleotide hybridises to complementary regions at the distal end ofthe nucleic acid strand to produce a 5′ overhang that contains thecoding sequence (codeC). The nucleic acid strand is then extended alongthe second identifier oligonucleotide using a polymerase. The secondidentifier oligonucleotide may be ligated to the nucleic acid strand andthe nucleic acid spacer strand. Coding information for all threechemical moieties in the pharmacophore is now encoded on the nucleicacid strand.

FIGS. 2D and 2E shows another alternative strategy for the production ofa three building block pharmacophore library in which the buildingblocks are coupled to both strands of the members.

A nucleic acid strand is first coupled to first and second chemicalmoieties (‘building blocks’). The nucleic acid strand contains a firstcoding sequence (codeA) encoding the first chemical moiety. A cleavablechimeric adaptor is used to couple an identifier oligonucleotide to thedistal end of the nucleic acid strand with respect to the moieties. Theidentifier oligonucleotide contains a second coding sequence (codeB)which encodes the identity of the second chemical moiety attached to thenucleic acid strand. The adaptor hybridizes to complementary bases onthe distal end of the nucleic acid strand and proximal end of theidentifier oligonucleotide forming a complex between identifieroligonucleotide, nucleic acid strand and adaptor. This brings an end ofthe identifier oligonucleotide into proximity to an end of the nucleicacid strand, such that ligation can occur under suitable conditions. Theends of the identifier oligonucleotide and nucleic acid strand are thenligated and the adaptor is removed.

A partner nucleic acid strand is coupled to a third chemical moiety. Thepartner nucleic acid strand contains a first spacer region (d-spacer) ata position corresponding to the first coding sequence (codeA) of thenucleic acid strand. The first spacer region does not hybridize to thefirst coding sequence (codeA).

A first cleavable chimeric adaptor is used to couple a nucleic acidspacer strand to the distal end of the partner nucleic acid strand withrespect to the third chemical moiety. The nucleic acid spacer strand iscapable of hybridizing to the nucleic acid strand and contains a secondspacer region (d-spacer II) at a position corresponding to the secondcoding sequence (codeB) of the nucleic acid strand. The second spacerregion does not hybridize to the coding sequence (codeB).

A second cleavable chimeric adaptor is used to couple a secondidentifier oligonucleotide to the distal end of the partner nucleic acidstrand with respect to the third chemical moiety (i.e. the secondidentifier oligonucleotide is coupled to the 5′ end of the nucleic acidspacer strand). The second identifier oligonucleotide contains a thirdcoding sequence (codeC) encoding the identity of the third chemicalmoiety. The first and second cleavable chimeric adaptors are thenremoved by purification, RNAse or pH to leave a partner nucleic acidstrand comprising a third coding sequence and first and second spacerregions at positions corresponding to the first and second codingsequences of the nucleic acid strand.

The nucleic acid strand and the partner nucleic acid strand are thenhybridized together through complementary regions in the strands to forma complex (FIG. 2E). The proximal end of the second identifieroligonucleotide of the partner strand with respect to the third chemicalmoiety hybridises to complementary regions at the distal end of thenucleic acid strand to produce a 5′ overhang in the complex thatcontains the third coding sequence (codeC). The nucleic acid strand isthen extended along the partner strand using a polymerase. Codinginformation for all three chemical moieties in the pharmacophore is nowencoded on the nucleic acid strand.

FIGS. 3A and 3B show strategies for the production of a three buildingblock pharmacophore library in which the building blocks are coupled toa single strand of the library members. A nucleic acid strand is coupledto a first chemical moiety. The nucleic acid strand is then contactedwith a cleavable adaptor and a first identifier oligonucleotide whichhybridize through complementary regions to form a trimeric complex. Thefirst identifier oligonucleotide contains a code sequence encoding theidentity of the first chemical moiety. The first identifieroligonucleotide is ligated to the nucleic acid strand and the adaptor iscleaved. A second chemical moiety is then coupled to the first chemicalmoiety. The nucleic acid strand is then contacted with a furthercleavable adaptor and a second identifier oligonucleotide whichhybridizes through complementary regions to form a complex. The secondidentifier oligonucleotide contains a code sequence encoding theidentity of the second chemical moiety. The second identifieroligonucleotide is ligated to the nucleic acid strand and the adaptor iscleaved. A third chemical moiety is then coupled to the first chemicalmoiety. In FIG. 3A, the nucleic acid strand is then contacted with afurther cleavable adaptor and a third identifier oligonucleotide whichhybridize through complementary regions to form a trimeric complex. Thethird identifier oligonucleotide contains a code sequence encoding theidentity of the third chemical moiety. The third identifieroligonucleotide is ligated to the nucleic acid strand and the adaptor iscleaved. The nucleic acid strand may than be combined with acomplementary sub-library to form a nucleic acid-encoded library. InFIG. 3B, the nucleic acid strand is then contacted with a thirdidentifier oligonucleotide which contains a code sequence encoding theidentity of the third chemical moiety. The third identifieroligonucleotide hybridizes through complementary regions to the 3′ endof the nucleic acid strand to form a complex with a single stranded 5′overhang comprising the code sequence. The nucleic acid strand is thenfilled in along the single stranded identifier oligonucleotide templateusing a polymerase such as a Klenow fragment to incorporate thecomplement of the code sequence. The nucleic acid strand may than becombined with a complementary sub-library to form a nucleic acid-encodedlibrary.

FIG. 4 shows analytical HPLC traces (recording absorbance at 260 nm and280 nm respectively) of A) untreated chimeric adapter and encodedligation oligonucleotide product of Table 2, B) high pH treatment withNaOH of the same oligonucleotides and C) RNase H treatment of the sameoligonucleotides.

FIG. 5 shows the results of polyacrylamide gel electrophoresis of the 5′coupled oligonucleotides and ligation products shown in Table 3 usingTBE Gel 49 (20% TBE) (FIG. 5A) and TBE Gel 50 (15% TBE Urea) (FIG. 5B).

FIG. 6 shows the results of polyacrylamide gel electrophoresis of the 3′coupled oligonucleotides and ligation products shown in Table 4 usingTBE Gel 57 (20% TBE) (FIG. 6A) and TBE Gel 58 (15% TBE Urea) (FIG. 6B).

EXPERIMENTS Example 1 Construction of a Sub-Library ofOligonucleotide-Compound Conjugates Using 3′-Aminomodified,5′-Phosphorylated Oligonucleotides

Synthetic oligonucleotides were purchased from various commercialsuppliers. They were stored as 1 mM and 100 μM stock solutions in at−20° C. Chemical compounds were purchased from various commercialsuppliers. Enzymes were purchased from various commercial suppliers.

1.1 Agarose and Polyacrylamide Gel Electrophoresis

DNA consisting of 10 to 300 nucleotides was analyzed on nativepolyacrylamide 20% TBE gels (1.0 mm, 12 well, Invitrogen) or ondenaturing polyacrylamide 15% TBE-Urea gels (1.0 mm, 12 well,Invitrogen). A current of 60 mA with a voltage of 180 V was applied for75 minutes on the electrophoresis box (Novex). The gels were stainedwith SYBR Green I. Preparative gel electrophoresis was performed on 2.0%agarose/TBE gels (stained with ethidium bromide) using 60 mA and 100 Vfor 25 minutes. SYBR Green I and ethidium bromide were detected by UVexcitation.

1.2 Synthesis of Fmoc-Protected Amino Acid and Carboxylic AcidOligonucleotide Conjugates

12.5 μl 100 mM Fmoc-protected amino acids or carboxylic acids (1.25 μmolin dry dimethyl sulfoxide [DMSO]) were activated for 30 min at 30° C.with 12 μl 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC,1.2 μmol) and 10 μl 333 mM N-hydroxysulfosuccinimide (S-NHS, 3.3 μmol,in DMSO/H2O, 2:1) in 215 μl dry DMSO and subsequently reacted overnightat 30° C. with 5 μl of amino-modified oligonucleotide (5 nmol) dissolvedin 50 μl 500 mM triethylamine/hydrogen chloride (TEA/HCl, 25 μmol,pH=10.0). Carboxylic acids were quenched with 20 μl 500 mM Tris/HCl(pH=8.1) at 30° C. for 1 h. Fmoc-protected amino acids were quenched andconcurrently deprotected with 5 μl 1 M tris(hydroxymethyl)aminomethane(Tris) and 5 μl pure TEA at 30° C. for 1 h. After quenching anddeprotection, the DNA-compound conjugate was precipitated with ethanol(see protocol for ethanol precipitation for compound-conjugates) beforepurifying by HPLC. The separated and collected compound conjugates werevacuum-dried overnight, redissolved in 100 μl H₂O, and analyzed byESI-MS.

1.3 Synthesis of Sulfonamide Oligonucleotide Conjugates

25 μl 100 mM sulfonyl chloride (2.5 μmol in dry acetonitrile [MeCN])were mixed with 25 μl 1 M sodium hydrogen carbonate in H2O (pH=9.0), 100μl MeCN, 95 μl H2O and subsequently reacted with 5 μl of theamino-modified oligonucleotide (5 nmol) overnight at 30° C. The reactionwas quenched with 20 μl 500 mM Tris/HCl (pH=8.1) at 30° C. for 1 h.After quenching the DNA-compound conjugate was precipitated with ethanol(see protocol for ethanol precipitation for compound-conjugates) beforepurification by HPLC. The separated and collected compound conjugateswere vacuum-dried overnight, redissolved in 100 μl H2O, and analyzed byESI-MS.

1.4 Synthesis of Oligonucleotide Conjugates from Carboxylic AcidAnhydrides

5.2 μl 100 mM carboxylic acid anhydrides (25 μl, 2.5 μmol in dry DMSO)were mixed together with 25 μl 500 mM sodium hydrogen phosphate in H2O(pH=7.1), 195 μl DMSO, 35 μl H2O and subsequently reacted with 5 μl ofthe amino-modified oligonucleotide (5 nmol) overnight at 30° C. Thereaction was quenched with 20 μl 500 mM Tris/HCl (pH=8.1) at 30° C. for1 h. After quenching, the DNA-compound conjugate was precipitated withethanol (see protocol for ethanol precipitation for compound-conjugates)before purifying by HPLC. The separated and collected compoundconjugates were vacuum-dried overnight, redissolved in 100 μl of H2O,and analyzed by ESI-MS.

1.5 Ethanol Precipitation of Compound-Oligonucleotide Conjugates

Before HPLC purification, the compound-olignucleotide conjugates wereprecipitated with ethanol. In this procedure, 100 μl 3 M sodium acetate(pH=4.7) and 30 μl 5 M acetic acid were added to the reactions. Aftervortexing, 1100 μl pure (100%) ethanol was added and the reactions wereallowed to stand for 30 min at 22° C. and 30 min at −20° C. beforecentrifugation (30 min, 13′200 rpm, 4° C.). Immediately aftercentrifugation, the supernatant was carefully discarded and the pelletwas dissolved in 500 μl 100 mM triethylammonium acetate (TEAA) buffer(pH=7.0) and subjected to HPLC purification.

1.6 High-Performance Liquid Chromatography (HPLC) ofOligonucleotide-Compound Conjugates

Oligonucleotide-conjugated compounds for the library were separated fromthe unreacted amino-modified Elib4.aT oligo by HPLC. A reverse-phasedC18-XTerra column (5 μm, 10×150 mm, Waters) with organic/inorganicparticle (silica and polymeric supports) was used as stationary phase.As a mobile phase, an aqueous, 100 mM triethylammonium acetate (TEAA)buffer C (pH=7.0) was used together with an acetonitrile gradient(buffer D: 100 mM TEAA in 80% MeCN/20% H2O. Depending on the retentiontime for a class of compounds, either a short (16 min, for morehydrophilic compounds) or a long (30 min, for more hydrophobic compoundsa gradient program was run (T=30° C., p=0-300 bar).

In order to distinguish oligonucleotides and oligonucleotide-conjugatesfrom starting compounds and side-products, absorption was monitored at260 nm and 280 nm. The oligonucleotide absorption ratio 260 nm/280 nm istypically 1.8/1. The collection of fractions was started after 4 minwith a minimum intensity threshold of 30′000 (106:=Abs=1 for theobserved channel [260 nm]). The minimum fraction collecting frame was 5s, the maximum 300 s.

1.7 Liquid Chromatography-Mass Spectrometry (LC-MS)

Mass-analysis of the oligo-coupled compounds was performed by thecombination of liquid chromatography with electrospray ionization massspectrometry (LC-ESI-MS). A reverse-phased C18-XBridge column (2.5 μm,2.1×50 mm, Waters) with organic/inorganic particle (silica and polymericsupports) was used as stationary phase. As a mobile phase, 400 mM1,1,1,3,3,3-hexafluoroisopropanol (HFIP), 2 mM triethylamin (TEA) bufferC was applied with a methanol gradient (buffer D: 400 mM HFIP, 2 mM TEAin 50% H2O/50% methanol (T=30° C., p=0-200 bar). A tandem-quadrupolemass spectrometer (Quattro micro API, Waters, Milford, Conn.) withelectrospray ionization (ESI) source was used for mass detection andanalysis. Mass spectrometric analyses were performed in negativeion-mode. ESI interface parameters were set as follows: disolvationtemperature: 200° C., source temperature: 110° C.; capillary voltage:3.0 kV; cone voltage: 40 V; scan time: 0.5 s; inter-scan delay time: 0.1s.

1.8 Encoding by Ligation

50 μl 2 μM compound-oligonucleotide conjugate (100 μmol), 10 μl 15 μMcoding oligonucleotide (150 pmol), 10 μl 30 μM chimeric RNA/DNA adapteroligonucleotide, 10 μl NEB 10× ligase buffer and 19.5 μl H2O were mixedand heated up to 90° C. for 2 min. Then the mixture was passively cooleddown to 22° C. (hybridization). Afterwards, 0.5 μl NEB ligase was added.Ligation was performed at 16° C. for 10 hours. The ligase wasinactivated for 15 min at 70° C.

1.9 Degradation of the Chimeric DNA/RNA Adapter

Hydrolysis of the RNA was achieved when an equivalent volume (13 μl) of200 mM sodium hydroxide and the ligation solution was mixed andincubated for 5 h at 22° C. The solution was then neutralized to pH=7.9.Alternatively, enzymatic cleavage was effectively carried out by adding5.3 μl of 10× RNase H reaction buffer, 33.7 H2O and 1.5 μl RNase H.RNase H was inactivated by heat denaturation (15 min, 70° C.).Optionally, the ligated oligonucleotide-compound conjugates could bepurified again by ethanol precipitation as described above. Equimolaramounts of encoded compounds were then mixed together to generate thedesired sub-library

Example 2 Construction of a Sub-Library of Oligonucleotide-CompoundConjugates Using 3′-Aminomodified, 5′-Phosphorylated Oligonucleotides2.1 Preparation of Amino-Modified Encoding Oligonucleotides

Amino-modified encoding oligonucleotides necessary were either purchasedfrom a commercial supplier or obtained by encoding by ligation: 50 μl 2μM amino-modified oligonucleotide (100 pmol), 10 μl 15 μM codingoligonucleotide (150 pmol), 10 μl 30 μM chimeric RNA/DNA adapteroligonucleotide, 10 μl NEB 10× ligase buffer and 19.5 μl H2O were mixedand heated up to 90° C. for 2 min. Then the mixture was passively cooleddown to 22° C. (hybridization). Afterwards, 0.5 μl NEB ligase was added.Ligation was performed at 16° C. for 10 hours. The ligase wasinactivated for 15 min at 70° C.

2.2 Degradation of the Chimeric DNA/RNA Adapter

Hydrolysis of the RNA was achieved when an equivalent volume (13 μl) of200 mM sodium hydroxide and the ligation solution was mixed andincubated for 5 h at 22° C. The solution was then neutralized to pH=7.9Alternatively, enzymatic cleavage was effectively carried out by adding5.3 μl of 10× RNase H reaction buffer, 33.7 H2O and 1.5 μl RNase H.RNase H was inactivated by heat denaturation (15 min, 70° C.).Optionally, the ligated oligonucleotide-compound conjugates could bepurified again by Ethanol precipitation as described above.

The sub-library of compound-oligonucleotide conjugates was then obtainedby chemically modifying the individual amino-modified encodedoligonuclotides, followed by Ethanol precipitation, HPLC purification,and MS-based analytics, as described in Example 1. Equimolar amounts ofencoded compounds were then mixed together to the desired sub-library.

Example 3 Construction of a Sub-Library of Oligonucleotide-CompoundConjugates Using 5′-Aminomodified Oligonucleotides

Commercially purchased oligonucleotides carrying a 5′ primary aminogroup and an individual encoding sequence were coupled to carboxylicacids, acyl chlorides, cyclic anhydrides, or isothiocyanates. Some ofthe carboxylic acids contained an Fmoc-protected amino group. Typically,for acyl chlorides, 200 μL of a 25 μM solution of oligonucleotide in 100mM NaHCO3, pH 9, was added to 200 μL of a 4 mM solution of acyl chloridein MeCN.

In the case of isothiocyanates, 100 μL of a 50 μM solution ofoligonucleotide in 100 mM KHPO4, pH 7.1, was added to 200 μL of a 2.6 mMsolution of isothiocyanate in DMSO. For cyclic anhydrides, 100 μL of a50 μM solution of oligonucleotide in 100 mM KHPO₄, pH 7.1, was added to200 μL of a 2.6 mM solution of anhydride in DMSO. To activate thecarboxylic acids, 22 μL of a solution containing 45 mM EDC and 180 mMsulfo-NHS in 15% H2O/85% DMSO was added to 230 μL of a 5.5 mM solutionof the carboxylic acid in DMSO. After 30 min at 30° C., 60 μL of asolution of 83 μM oligonucleotide in 420 mM TEA/HCl, pH 10, was added.All reactions were stirred for 12 h at 30° C. The reactions werequenched by adding 20 μL of 500 mM Tris/HCl, pH 8, and stirred for anadditional 1 h at 30° C. In the case of Fmoc-protected compounds, thequenching and removal of the Fmoc group was performed by addition of 5μL of 1 M Tris and 5 μL of triethylamine and stirring for 1 h at 30° C.

For HPLC purification, 400 μL of 100 mM TEAA, pH 7, was added to thereaction mixture. In the case of the Fmoc samples, 20 μL of 1 M HCl wasadditionally added. Purifications were performed by HPLC on an XTerraPrep RP18 column (5 μM, 10×150 mm) using a linear gradient from 10 to40% MeCN in 100 mM TEAA. The desired samples were redissolved in 100 μLof H2O. An amount of 5 μL was analyzed by LC-ESI-MS on an XTerra RP18column (5 μM, 4.6×20 mm) using a linear gradient from 0 to 50% MeOH over1 min in 400 mM HFIP/5 mM TEA. The mass spectrum was measured from 900to 2000 m/z by a Waters Quattro Micro instrument. The mass spectra ofoligonucleotides before and after conjugation were analyzed. The samplescontaining the oligonucleotide-compound conjugates of the expected sizewere pooled and precipitated by adding 10% v/v of 3 M NaAc, pH4.7, and250% v/v of EtOH. The pellets were collected by centrifugation andwashed by addition of ice cold 85% EtOH, followed by drying undervacuum. The oligonucleotide-compound conjugates were then redissolved in100 μL of H2O, and the OD260 was determined by a ND-1000 (Nanodrop).Equimolar amounts of encoded compounds were then mixed together togenerate the desired sub-library.

Example 4 Construction of a Sub-Library Displaying Two Chemical BuildingBlocks (2BB) Using 5′-Aminomodified Oligonucleotides 4.1 DNA-Conjugationof Carboxylic Acids as First Building Block (BB1)

Protected DNA 45-mers with a terminal 5′-amino modifier C12 attached tothe solid support (controlled pore glass) were distributed intosynthesis cartridges (approx. 50 nmol). The supports were washed withMeCN and DCM (2×). A solution of 3% trichloroacetic in DCM (1-2 mL) wasdropwise eluted from the cartridge followed by washing with DCM (2 mL)and these two steps were repeated 5 times. The solid support was washedwith DCM (1×1 mL) and MeCN (2×1 mL). The solid support was treated witha solution of Fmoc-L-DAP(Mtt)-OH (50 mM), HATU (50 mM) and DIEA (150 mM)in DMF (0.5 mL) and let react for 2 h at room temperature. The solutionwas removed and the solid support rinsed with DMF (2×1 mL), MeCN (1×1mL) and DCM (2×1 mL). The Mtt-group was removed as described above forthe Mmt-group. The solid support was then treated with a solution of thecorresponding carboxylic acid (50 mM), HATU (50 mM) and DIEA (150 mM) inDMF (0.5 mL) and let react overnight. The solution was removed and thesupport rinsed with DMF (2×1 mL), MeCN (2×1 mL) and dried under a streamof air. The DNA was cleaved from the solid support and deprotected by 2h incubation in conc. aq. NH₃/MeNH₂ (AMA) (1 mL) at room temperature.The AMA solution was evaporated, the residue dissolved in water (0.2 mL)and the DNA conjugates purified by reverse-phase HPLC.Product-containing fractions were combined, evaporated and analyzed byLC-MS measurement.

4.2 DNA-Conjugation of Carboxylic Acids as Second Building Block (BB2)

Equimolar amounts of the DNA-conjugates obtained as described above werecombined and further derivatized: The combined conjugates (0.75 nmol)were immobilized on DEAE sepharose (0.1 mL of slurry). The resin waswashed with 10 mM aq. AcOH (2×0.5 mL), water (2×0.5 mL) and DMSO (2×0.5mL). To the resin-immobilized DNA was added a solution of thecorresponding carboxylic acid (50 mM), EDC (50 mM) and HOAt (5 mM) inDMSO (0.5 mL). The slurry was agitated for 2 h at room temperature. Thesolution was removed and the resin washed with DMSO (1×0.5 mL) andtreated with freshly activated reaction solution. These steps wererepeated to reach three coupling steps of 2 h each. The reactionsolution was removed and the resin washed with DMSO (2×0.5 mL) and 10 mMaq. AcOH (3×0.5 mL). The DNA was eluted from the resin by incubationwith 3 M AcOH buffer (pH 4.75) for 5 min. The DNA-conjugates wereisolated by ethanol-precipitation and the pellets redissolved indeionized water (50 μL). To ensure a high degree of conversion forchemical BB2, all used carboxylic acids were tested for couplingefficiency and only carboxylic acids with high conversion yields in testreactions (typically >80%) were used for library synthesis. Theindividual DNA-chemical conjugates constitute a (not pooled) sub-librarywhich is encoded for BB1 but not yet for BB2 and can be used as startingmaterial for the library construction described in Examples 6-8.

Example 5 Preparation of a DNA-Encoded Library [1+1 Library (FIG.1A+1B)]

20 μl 0.5 μM of pooled 3′-compound oligonucleotide conjugates (e.g.sub-library of Example 1 or 2), 1 μl 10 μM of pooled 5′-compoundoligonucleotide conjugates (e.g. sub-library of Example 3), 10 μl10×NEB2 reaction buffer, 57 μl H₂O and 8 μl 500 μM dNTPs were mixed andheated up to 90° C. for 2 min, then cooled to 22° C. for hybridization.2 μl NEB Klenow polymerase was added and the sample was incubated at 25°C. for 90 min, optionally followed by a purification step. The obtainedencoded self-assembling chemical library could optionally be stored ordirectly used for target-based selections.

Example 6 Preparation of a DNA-Encoded Library [2+1 Library (FIG. 2A)]

The individual sub-library members of Example 3, which carry thechemical building blocks BB1 and BB2 and which are encoded for BB1 (butnot yet for BB2) were encoded for BB2 according to the followingprocedure:

6.1 Encoding by Ligation

50 μl of 2 μM compound-oligonucleotide conjugate (100 pmol), 10 μl 15 μMcoding oligonucleotide (150 pmol), 10 μl 30 μM chimeric RNA/DNA adapteroligonucleotide, 10 μl NEB 10× ligase buffer and 19.5 μl H₂O were mixedand heated up to 90° C. for 2 min. Then the mixture was passively cooleddown to 22° C. (hybridization). Afterwards, 0.5 μl NEB ligase was added.Ligation was performed at 16° C. for 10 hours. The ligase wasinactivated for 15 min at 70° C.

6.2 Degradation of the Chimeric DNA/RNA Adapter

Hydrolysis of the RNA was achieved when an equivalent volume (13 μl) of200 mM sodium hydroxide and the ligation solution was mixed andincubated for 5 h at 22° C. The solution was then neutralized to pH-7.9.Alternatively, enzymatic cleavage was effectively carried out by adding5.3 μl of 10× RNase H reaction buffer, 33.7 H2O and 1.5 μl RNase H.RNase H was inactivated by heat denaturation (15 min, 70° C.).Optionally, the ligated oligonucleotide-compound conjugates could bepurified again by Ethanol precipitation as described above.

Equimolar amounts of encoded compounds were then mixed together to thedesired sub-library A. A portion of sub-library A was then split into200 vials (10 μl of 20 nM compound-oligonucleotide conjugates) and eachvial contained: 10 μl of 20 nM individual sub-library B member, 10 μl of20 nM DNA/RNA adaptor oligonucleotide (d-spacerII) and 10 μl of 20 nMindividual coding oligonucleotide (code C), 10 μl NEB 10× ligase bufferand 10 μl H2O. The solutions were mixed and heated up to 90° C. for 2min. Then the mixture was cooled down to 22° C. (hybridization).Afterwards, 0.5 μl NEB ligase was added. Ligation was performed at 16°C. for 10 hours. Equimolar amounts of the 200 vials were mixed together,optionally followed by a purification step. The obtained DNA-encodedchemical library could optionally be stored or directly used fortarget-based selections.

Example 7 Preparation of a DNA-Encoded Library [2+1 Library (FIG. 2B)]

The individual sub-library members of Example 3, which carry thechemical building blocks BB1 and BB2 and which are encoded for BB1 (butnot yet for BB2) were encoded for BB2 according to the followingprocedure:

7.1 Encoding by Ligation

50 μl of 2 μM compound-oligonucleotide conjugate (100 pmol), 10 μl 15 μMcoding oligonucleotide (150 pmol), 10 μl 30 μM adapter oligonucleotidecontaining 2 abasic sites, 10 μl NEB 10× ligase buffer and 19.5 μl H2Owere mixed and heated up to 90° C. for 2 min. Then the mixture waspassively cooled down to 22° C. (hybridization). Afterwards, 0.5 μl NEBligase was added. Ligation was performed at 16° C. for 10 hours. Theligase was inactivated for 15 min at 70° C. Optionally, the ligatedoligonucleotide-compound conjugates could be purified as describedabove.

Equimolar amounts of encoded compounds were then mixed together to thedesired sub-library A. A portion of sub-library A was then split into200 vials (10 μl of 20 nM compound-oligonucleotide conjugates) and eachvial contained: 10 μl of 20 nM individual sub-library B member, 10 μl of20 nM adapter oligonucleotide containing 2 abasic sites and 10 μl of 20nM individual coding oligonucleotide (code C), 10 μl NEB 10× ligasebuffer and 10 μl H₂O. The solutions were mixed and heated up to 90° C.for 2 min. Then the mixture was cooled down to 22° C. (hybridization).Afterwards, 0.5 μl NEB ligase was added. Ligation was performed at 16°C. for 10 hours. Equimolar amounts of the 200 vials were mixed together,optionally followed by a purification step. The obtained DNA-encodedchemical library could optionally be stored or directly used fortarget-based selections.

Example 8 Preparation of a DNA-Encoded Library [2+1 Library (FIG. 2C)]

The individual sub-library members of Example 3, which carry thechemical building blocks BB1 and BB2 and which are encoded for BB1 (butnot yet for BB2) were encoded for BB2 according to the followingprocedure.

8.1 Encoding by Ligation

50 μl of 2 μM compound-oligonucleotide conjugate (100 pmol), 10 μl 15 μMcoding oligonucleotide (150 pmol), 10 μl 30 μM chimeric RNA/DNA adapteroligonucleotide, 10 μl NEB 10× ligase buffer and 19.5 μl H₂O were mixedand heated up to 90° C. for 2 min. Then the mixture was passively cooleddown to 22° C. (hybridization). Afterwards, 0.5 μl NEB ligase was added.Ligation was performed at 16° C. for 10 hours. The ligase wasinactivated for 15 min at 70° C.

8.2 Degradation of the Chimeric DNA/RNA Adapter

Hydrolysis of the RNA was achieved when an equivalent volume (13 μl) of200 mM sodium hydroxide and the ligation solution was mixed andincubated for 5 h at 22° C. The solution was then neutralized to pH=7.9.Alternatively, enzymatic cleavage was effectively carried out by adding5.3 μl of 10× RNase H reaction buffer, 33.7 H₂O and 1.5 μl RNase H.RNase H was inactivated by heat denaturation (15 min, 70° C.).Optionally, the ligated oligonucleotide-compound conjugates could bepurified again by Ethanol precipitation as described above.

Equimolar amounts of encoded compounds were then mixed together to thedesired sub-library A. A portion of sub-library A was then split into200 vials (10 μl of 20 nM compound-oligonucleotide conjugates) and eachvial contained: 10 μl of 20 nM individual sub-library B member, 10 μl of20 nM DNA/RNA adaptor oligonucleotide (d-spacerII) and 10 μl of 20 nMindividual coding oligonucleotide (code C), 10 μl 10×NEB2 reactionbuffer, 52 μl H2O and 8 μl 500 μM dNTPs were mixed and heated up to 90°C. for 2 min, then cooled to 22° C. for hybridization. 2 μl Klenowpolymerase was added and the sample was incubated at 25° C. for 90 min,optionally followed by a purification step. Equimolar amounts of the 200vials were mixed together, optionally followed by a purification step.The obtained DNA-encoded chemical library could optionally be stored ordirectly used for target-based selections.

Example 9 Chimeric Adaptors

Chimeric adapters were used to facilitate the ligation mediated by T4DNA ligase, as this enzyme only seals nicks in double stranded DNA.Chimeric adapters were required for the enzymatic reaction but needed tobe disposed of afterwards. The Chimeric adapters were DNAoligonucleotides with intermittent RNA nucleotides. The adapter-specificdisintegration was achieved by NaOH-treatment of the ligation products,which cleaves the chimeric adapters at the RNA sites. An alternativedisintegration strategy is the cleavage using RNase H. For the 2+1library of Example 10, the three Chimeric Adapters shown in Table 1 wereemployed.

9.1 Degradation Tests

The Chimeric Adapters shown in Table 2 were tested for degradation bymeans of NaOH treatment (high pH) or RNase H treatment. FIG. 4 showsanalytical HPLC traces (recording absorbance at 260 nm and 280 nmrespectively) of a) untreated chimeric adapter and encoded ligationoligonucleotide product, b) high pH treatment with NaOH of the sameoligonucleotides and c) RNase H treatment of the same oligonucleotides.Both methods show disintegration of the DNA/RNA chimeric adapteroligonucleotide.

9.3 Ligation

The ligation of nucleic acid strands carrying compounds at their 5′ endusing chimeric adapters was assessed. TBE and TBE-Urea gels (lifetechnologies, Novex TBE Gels, 20%, 15 well, Cat. No. EC63155BOX; lifetechnologies, Novex® TBE-Urea Gels, 15%, 15 well, Cat. No. EC68855BOX),were loaded as shown in Table 3 and subjected to electrophoresis. Theresults are shown in FIG. 5 and indicate that strands were successfullyligated and the chimeric adapters removed by standard purificationtechniques.

The ligation of nucleic acid strands carrying compounds at their 3′ endusing chimeric adapters was assessed. TBE and TBE-Urea gels (lifetechnologies, Novex TBE Gels, 20%, 15 well, Cat. No. EC63155BOX; lifetechnologies, Novex® TBE-Urea Gels, 15%, 15 well, Cat. No. EC68855BOX),were loaded as shown in Table 4 and subjected to electrophoresis. Theresults are shown in FIG. 6 and indicate that strands were successfullyligated and the chimeric adapters removed by standard purificationtechniques.

Example 10 Preparation of a DNA-Encoded Library [2+1 Library (FIGS. 2Dand 2E)]

The ESAC 2+1 library consists of two sub-libraries. The 5′-sub-librarycarries two compounds at the 5′-end of a single-stranded oligonucleotidewhile the 3′-sub-library consists of one compound, coupled to the 3′-endof a complementary single-stranded oligonucleotide. Both sub-library aremixed in equimolar amounts and hybridized by heating. Klenow fill-in isused to transfer coding information from the 3′-strand to the 5′-strand.

10.1 5′-Sublibrary (2 Building Blocks)

The 5′-Sublibrary was generated in split-and-pool fashion. Buildingblock 1 was coupled to an oligonucleotide that contains Code 1.Compound-oligonucleotide conjugates were pooled, split to equimolaramounts and coupled to building block 2. These intermediate librarymembers were encoded via ligation: conjugates were incubated with anequimolar amount of an oligonucleotide that contained code two and anexcess of a chimeric adapter oligonucleotide (DNA/RNA hybrid) (see Table5). The Code 1 and Code 2 oligonucleotides were ligated using T4 DNALigase. The chimeric adapter was disintegrated using 250 mM NaOH.Finally, the ligation product was purified using the Qiagen QIAquick gelextraction kit and library members were pooled again in equimolaramounts in order to yield the final 5′-sublibrary.

10.2 3′-Sub-Library

The 3′-sub-library carries building block 3 at the 3′-end of asingle-stranded oligonucleotide. The 3′-oligonucleotide, named Elib4.aT,contains a d-spacer (abasic nucleotide backbone) in order to allowhybridization to Code 1. Elib4.aT was ligated (as described above) to asecond d spacer that allowed hybridization to Code 2, and purified asdescribed above (see Table 6). The oligonucleotide containing Code 2 wasadded in a second ligation step. The final product was purified andpooled in equimolar amounts in order to yield the final 3′-sublibrary.

10.2. Hybridization and Klenow Fill-In

The 3′- and 5′-sublibraries were mixed in equimolar amounts. Heating andsubsequent cooling down to room temperature leads to the hybridization(=combinatorial assembly) of the two sub-libraries. Klenow polymerasewas used to fill in the Code 3 information of the 3′-strand to the5′-strand as shown in Table 7, which was amplified by PCR.

Example 11 Preparation of a DNA-Encoded Library of Three or MoreBuilding Blocks [(FIG. 3A)]

5′-Amino-modified oligonucleotides were modified with a first chemicalbuilding block as described in Examples 1-3 (i.e. in liquid or on solidphase). The compound-oligonucleotide conjugates were then purified andindividually ligated with an encoding oligonucleotide, by the help of aRNA/DNA adaptor oligonucleotide, as described in Examples 1-3. Theadaptor molecules were then removed by either pH-based cleavage or RNAseH addition, optionally followed by a purification step, described inExamples 1-3. The obtained encoded compound-oligonucleotide conjugateswere pooled in equimolar amounts and then split into a set of b vials,for the modification with b building block 2 (BB2) compounds.

The couplings were performed either in solution or while the DNA wasattached to a solid support, as described in Examples 1-3. The b poolsof compound-oligonucleotide conjugates were then individually ligatedwith an encoding oligonucleotide, by the help of a RNA/DNA adaptoroligonucleotide, as described in Examples 1-3. The adaptor moleculeswere then removed by either pH-based cleavage or RNAse H addition,optionally followed by a purification step, described in Examples 1-3.The obtained set of encoded compound-oligonucleotide conjugates werepooled in equimolar amounts and then split into a set of c vials, forthe modification with c building block 3 (BB3) compounds. The couplingswere performed either in solution or while the DNA was attached to asolid support, as described in Examples 1-3. The b pools ofcompound-oligonucleotide conjugates were then individually ligated withan encoding oligonucleotide, by the help of a RNA/DNA adaptoroligonucleotide, as described in Examples 1-3. The adaptor moleculeswere then removed by either pH-based cleavage or RNAse H addition,optionally followed by a purification step, described in Examples 1-3.The obtained set of encoded compound-oligonucleotide conjugates carryingsets of 3 encoded building blocks were either submitted to furtherrounds of for the modification with further sets of building blocksfollowed by encoding or mixed together to the desired sub-library.Optionally, using a suitable DNA polymerase the sub-library wasconverted into a double stranded DNA-encoded chemical library, whichcould optionally be stored or directly used for target-based selections.

Example 12 Preparation of a DNA-Encoded Library [(FIG. 3B)]

5′-Amino-modified oligonucleotides were modified with a first chemicalbuilding block as described in Examples 1-3 (i.e. in liquid or on solidphase). The compound-oligonucleotide conjugates were then purified andindividually ligated with an encoding oligonucleotide, by the help of aRNA/DNA adaptor oligonucleotide, as described in Examples 1-3. Theadaptor molecules were then removed by either pH-based cleavage or RNAseH addition, optionally followed by a purification step, described inExamples 1-3. The obtained encoded compound-oligonucleotide conjugateswere pooled in equimolar amounts and then split into a set of b vials,for the modification with b building block 2 (BB2) compounds. Thecouplings were performed either in solution or while the DNA wasattached to a solid support, as described in Examples 1-3. The b poolsof compound-oligonucleotide conjugates were then individually ligatedwith an encoding oligonucleotide, by the help of a RNA/DNA adaptoroligonucleotide, as described in Examples 1-3. The adaptor moleculeswere then removed either by pH-based cleavage or RNAse H cleavage,optionally followed by a purification step, described in Examples 1-3.The obtained sets of encoded compound-oligonucleotide conjugates werepooled in equimolar amounts and conjugation with further n sets ofbuilding blocks (n>1) was performed as described before. The ultimateencoding step was not performed by ligation but by polymerase-mediatedfill-in. In this case, a fill-in reaction with an encodingoligonucleotide complementary to a sequence between the (n−1)^(th) codeand the 3′ terminus of the compound-oligonucleotide conjugate strand wasperformed, as described in Examples 5 and 6, leading to adouble-stranded DNA-encoded chemical library, which could optionally bestored or directly used for target-based selections.

Example 13 Affinity Screening of a DNA-Encoded Chemical Library Againsta Target Protein of Interest

Affinity selections were performed using a Thermo Scientific KingFishermagnetic particle processor. Streptavidin-coated magnetic beads (0.1 mg)were resuspended in 100 μl PBS (50 mM NaPi, 100 mM NaCl, pH 7.4) andsubsequently incubated with 100 μl biotinylated target protein ofinterest (0.1 μM/1.0 μM concentration) for 30 min with continuous gentlemixing. target protein-coated beads were washed three times with 200 μlPBST (50 mM NaPi, 100 mM NaCl, 0.05% (v/v) Tween-20, pH 7.4) that wassupplemented with 100 μM biotin in order to block remaining bindingsites on Streptavidin, and subsequently incubated with 100 μl of theDNA-encoded chemical library (100 nM total concentration, in PBST) for 1h with continuous gentle mixing. After removing unbound library membersby washing with 200 μl PBST for five times, beads carrying bound librarymembers were resuspended in 100 μl buffer EB (QIAGEN) and the DNAcompound conjugates were separated from the beads by heat denaturationof Streptavidin and target protein (95° C. for 5 min).

REFERENCES

-   1 Mannocci, L. et al. PNAS USA 105(46):17670-17675-   2 Brenner, S. and Lerner, R. A. PNAS USA 89 (1992), 5381-5383-   3 Nielsen, J., et al., J. Am. Chem. Soc. 115 (1993)-   4 Needels et al., M. C., PNAS USA 90 (1993), 10700-10704-   5 Gartner, Z. J., et al., Science 305 (2004), 1601-1605-   6 Melkko, S., et al., Nat. Biotechnol. 22 568-574 (2004)-   7 Sprinz, K. I., et al., Bioorg. Med. Chem. Lett. 15 (2005), pp.    3908-3911-   8 Leimbacher et al Chemistry. 2012 Jun. 18; 18(25):7729-37-   9 Clark et al Nat Chem Biol. 2009 September; 5(9):647-54

TABLE 1 Adapter 5′-CGTC

ATCCG

CGCCAT

GGACTCG-3′ Adapter G1 5′-CGA

TCCCAT

GCGCCG

ATCGACG-3′ Adapter G2 5′-GCCTC

AGGCGT

ATCCTAC-3′

 RNA nucleotides 

TABLE 2 Test Adapter 5′-CGA

CATGGC

CTGC-3′

 RNA nucleotide Test Ligation Product5′-CCTGCATCGAATGGATCCGTGXXXXXXXXGCAGCTGCGCCATGGGACTCGddddddCAGCACACAGAATTCAGAAGCTCC-3′

TABLE 3 lane 1    Code 1 (45 nt)5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGC-3′ lane 2   Code 2 5′P (27 nt) 5′-CGGATCGACGYYYYYYYGCCTCGAGGC-3′ lane 3   Adapter (25 nt) 3′-GCTCAGGGTACCGCGGCCTAGCTGC-5′ lane 4 hybridization   Code 1 (45 nt) 5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTNICK:     Code 2 5′P (27 nt) CCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′3′-GCTCAGGGTACCGCGGCCTAGCTGC-5′    Adapter (25 nt) lane 5 ligation   Code 1 + Code 2 (72 nt)5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′ 3′-GCTCAGGGTACCGCGGCCTAGCTGC-5′   Adapter (25 nt) lanes 6-9 purified ligation    Code 1 +Code 2 (72 nt) 5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′ lane 6 QIA nucleotide removal kit NaOH -lane 7 QIA nucleotide removal kit NaOH + lane 8 QIA gel extraction kitNaOh - lane 9 QIA gel extraction kit NaOH +

TABLE 4 lane 1    Code 3′ (31 nt) 3′-CACTAGGATGzzzzzzCGCGGTACCCTGAGC-5′lane 2    d-spacer 2 (31 nt) 3′-CGCGGCCTAGCTGCdddddddCGGAGCTCCG-5′lane 3    Adapter G2 (20 nt) 5′-GCCTCGAGGCGTGATCCTAC-3′lane 4 hybridication    Adapter G2 (20 nt) 5′-GCCTCGAGGCGTGATCCTAC-3′3′-CGCGGCCTAGCTGCddddddd    d-spacer 2 (31 nt)CGGAGCTCCGCACTAGGATGzzzzzzCGCGGTACCCTGAGC-5′ NICK: Code 3′ (31 nt)lane 5 ligation    Adapter G2 (20 nt) 5′-GCCTCGAGGCGTGATCCTAC-3′3′-CGCGGCCTAGCTGCddddddd    d-spacer 2 + Code 3′ (62 nt)CGGAGCTCCGCACTAGGATGzzzzzzCGCGGTACCCTGAGC-5′lanes 6-11 purified ligation    d-spacer 2 + Code 3′ (62 nt)3′-CGCGGCCTAGCTGCddddddd CGGAGCTCCGCACTAGGATGzzzzzzCGCGGTACCCTGAGC-5′lane 6 QIA PCR purification kit NaOH - lane 7 QIA nucleotide removal kitNaOH - lane 8 QIA gel extr kit with isopropanol NaOH - lane 9QIA gel extr kit no isopropanol NaOH - lane 10 MN NTI NaOH - lane 11MN NTC NaOH -

TABLE 5 Step 1: Ligate Code 1 + Code 2 (T4 DNA Ligase)    Code 1 (45 nt)5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGC-3′   Code 2 5′P (27 nt) 5′-CGGATCGACGYYYYYYYGCCTCGAGGC-3′3′-GCTCAGGGTACCGCGGCCTAGCTGC-5′    Adapter (25 nt)    Code 1 +Code 2 (72 nt) 5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′ 3′-GCTCAGGGTACCGCGGCCTAGCTGC-5′   Adapter (25 nt) Step 2: Disintegration of chimeric Adapter byNaOH treatment Step 3: Purification using spin columns    Code 1 +Code 2 (72 nt) 5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′ XXXXXX 6 variable nucleotides of Code 1YYYYYYY 7 variable nucleotides of Code 2 zzzzzz 6 complementary variablenucleotides of Code 3 ZZZZZZ 6 variable nucleotides of Code 3 ddddddabasic nucleotide backbone P phophate nt nucleotides

TABLE 6 Step 1: Ligate Elib4.aT + d-spacer 2 (T4 DNA Ligase)   Adapter G1 (25 nt) 5′-CGAGTCCCATGGCGCCGGATCGACG-3′3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTAC-5′    Elib4.aT (41 nt)3′-CGCGGCCTAGCTGCdddddddCGGAGCTCCG-5′    d-spacer 2 (31 nt)   Adapter G1 (25 nt) 5′-CGAGTCCCATGGCGCCGGATCGACG-3′3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGCGGCC    Elib4.aT +d-spacer 2 (72 nt) TAGCTGCdddddddCGGAGCTCCG-5′Step 2: Disintegration of chimeric Adapter by NaOH treatmentStep 3: Purification using spin columns3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGCGGCC    Elib4.aT +d-spacer 2 (72 nt) TAGCTGCdddddddCGGAGCTCCG-5′Step 4: Ligate Elib4.aT/d-spacer 2 + Code 3′ (T4 DNA Ligase)   Adapter G2 (20 nt) 5′-GCCTCGAGGCGTGATCCTAC-3′3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGCGGCC    Elib4.aT +d-spacer 2 (72 nt) TAGCTGCdddddddCGGAGCTCCG-5′3′-CACTAGGATGzzzzzzCGCGGTACCCTGAGC-5′    Code 3′ (31 nt)   Adapter G2 (20 nt) 5′-GCCTCGAGGCGTGATCCTAC-3′3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGCGGCC    Elib4.at +d-spacer 2 + Code 3′ (103 nt)TAGCTGCdddddddCGGAGCTCCGCACTAGGATGzzzzzzCGCGGTACCCT GAGC-5′Step 5: Disintegration of chimeric Adapter by NaOH treatmentStep 6: Purification using spin columns3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGCGGCC   Elib4 intermediate library + d-spacer 2 +    Code 3′ (103 nt)TAGCTGCdddddddCGGAGCTCCGCACTAGGATGzzzzzzCGCGGTACCCT GAGC-5′

TABLE 7 Step 1: Hybridization of Sub-Libraries    Code 1 +Code 2 (72 nt) 5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGC    Elib4.aT +d-spacer 2 + Code 3′ (103 nt)GGCCTAGCTGCdddddddCGGAGCTCCGCACTAGGATGzzzzzzCGC GGTACCCTGAGC-5′step 2: Klenow Polymerase fill-in    Code 1 + Code 2 (72 nt)5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGC-3′ --->3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGC    Elib4.aT +d-spacer 2 + Code 3′ (103 nt)GGCCTAGCTGCdddddddCGGAGCTCCGCACTAGGATGzzzzzzCGC GGTACCCTGAGC-5′   Code 1 + Code 2 + Code 3 (103 nt)5′-GGAGCTTCTGAATTCTGTGTGCTGXXXXXXCGAGTCCCATGGCGCCGGATCGACGYYYYYYYGCCTCGAGGCGTGATCCTACZZZZZZGCG CCATGGGACTCG-3′3′-CCTCGAAGACTTAAGACACACGACddddddGCTCAGGGTACCGC    Elib4.aT +d-spacer 2 + Code 3′ (103 nt)GGCCTAGCTGCdddddddCGGAGCTCCGCACTAGGATGzzzzzzCGC GGTACCCTGAGC-5′Final library. Ready to use.

1.-40. (canceled)
 41. A method of producing a nucleic acid encoded chemical sub-library comprising; (i) providing a population of first nucleic acid strands, each nucleic acid strand being coupled or couplable to a member of a diverse population of chemical moieties, (ii) contacting the nucleic acid strands with identifier oligonucleotides comprising a coding sequence and one or more adaptor oligonucleotides, such that the one or more adaptor oligonucleotides hybridize to the nucleic acid strands and the identifier oligonucleotides to form a partially double-stranded complex, wherein each nucleic acid strand is contacted with an identifier oligonucleotide comprising a coding sequence that encodes a chemical moiety that is coupled or couplable to the nucleic acid strand, and; wherein each of said one or more adaptor oligonucleotides hybridizes to more than one nucleic acid strand in the population and more than one different identifier oligonucleotide, (iii) ligating the nucleic acid strands to the identifier oligonucleotides in the partially double-stranded complexes, such that the identifier oligonucleotides are incorporated into the nucleic acid strands and (iv) cleaving the adaptor oligonucleotides to remove them from the nucleic acid strands and the identifier oligonucleotides, thereby producing a sub-library comprising nucleic acid strands coupled or couplable to a member of a diverse population of chemical moieties, wherein each nucleic acid strand comprises a coding sequence that encodes the chemical moiety that is coupled to the nucleic acid strand.
 42. A method according to claim 41 wherein the adaptor oligonucleotide comprises one or more ribonucleotide bases.
 43. A method of producing a nucleic acid encoded chemical library comprising; (i) producing a sub-library according to a method comprising; (a) providing a population of first nucleic acid strands, each nucleic acid strand being coupled or couplable to a member of a diverse population of chemical moieties and comprising a non-hybridisable spacer, (b) contacting the nucleic acid strands with identifier oligonucleotides comprising a coding sequence and one or more adaptor oligonucleotides, such that the one or more adaptor oligonucleotides hybridize to the nucleic acid strands and the identifier oligonucleotides to form a partially double-stranded complex, wherein each nucleic acid strand is contacted with an identifier oligonucleotide comprising a coding sequence that encodes a chemical moiety that is coupled or couplable to the nucleic acid strand, and; wherein each of said one or more adaptor oligonucleotides hybridizes to more than one nucleic acid strand in the population and more than one different identifier oligonucleotide, (c) ligating the nucleic acid strands to the identifier oligonucleotides in the partially double-stranded complexes, such that the identifier oligonucleotides are incorporated into the nucleic acid strands, thereby producing a sub-library comprising first nucleic acid strands coupled or couplable to a member of a diverse population of chemical moieties, wherein each first nucleic acid strand comprises a coding sequence that encodes the chemical moiety that is coupled to the nucleic acid strand and the non-hybridisable spacer; (ii) hybridizing the first nucleic acid strands to second nucleic acid strands to form double-stranded complexes, wherein the second nucleic acid strands are coupled to a second diverse population of chemical moieties, each second nucleic acid strand comprising a second coding sequence that encodes the chemical moiety that is coupled to it, the position of the second coding sequence in the second nucleic acid strands corresponding in the double-stranded complex to the position of the spacer in the first nucleic acid strands in the double-stranded complexes, such that the second coding sequences do not preferentially hybridise to the first nucleic acid strands, and (iii) extending the second nucleic acid strands along the nucleic acid strands to produce a library comprising members having a double strand nucleic acid molecule comprising the first and second nucleic acid strands; the first diverse population of chemical moieties being coupled to the first nucleic acid strands and the second diverse population of chemical moieties being coupled to the second nucleic acid strands, said chemical moieties form pharmacophores in the library members, wherein the second nucleic acid strand comprises a first coding sequence that encodes the chemical moiety from the first diverse population and a second coding sequence that encodes the chemical moiety from the second diverse population.
 44. A method according to claim 43 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 45. A method according to claim 43 wherein the adaptor oligonucleotide is removed after the ligation by cleaving the adaptor oligonucleotide.
 46. A method according to claim 45 wherein the adaptor oligonucleotide comprises one or more ribonucleotide bases.
 47. A method according to claim 41 comprising; (v) coupling a diverse population of second chemical moieties to the first nucleic acid strands, (vi) contacting the first nucleic acid strands coupled to the second chemical moieties with a second adaptor oligonucleotide and a second identifier oligonucleotide comprising a coding sequence, such that the second adaptor hybridizes to the nucleic acid strands and the identifier oligonucleotides to form partially double-stranded complexes, wherein each nucleic acid strand is contacted with a second identifier oligonucleotide that comprises a second coding sequence that encodes the second chemical moiety that is coupled to the nucleic acid strand, and; wherein all the nucleic acid strands are contacted with the identical adaptor oligonucleotides, and (vii) ligating the nucleic acid strands to the second identifier oligonucleotides in the double-stranded complexes, such that the second coding sequence identifier oligonucleotides are incorporated into the nucleic acid strands.
 48. A method according to claim 47 comprising cleaving the second adaptor oligonucleotide to remove it from the nucleic acid strands and the second identifier oligonucleotides, optionally wherein the second adaptor oligonucleotide comprises one or more ribonucleotide bases.
 49. A method according to claim 47 further comprising; (viii) coupling a diverse population of further chemical moieties to the first nucleic acid strands, (ix) contacting the first nucleic acid strands coupled to the further chemical moieties with a further adaptor and a further identifier oligonucleotide comprising a coding sequence, such that the further adaptor hybridizes to the nucleic acid strands and the identifier oligonucleotides to form partially double-stranded complexes, wherein each nucleic acid strand is contacted with a further identifier oligonucleotide that comprises a further coding sequence that encodes the further chemical moiety that is coupled to the nucleic acid strand, and; wherein all the first nucleic acid strands are contacted with the same adaptor oligonucleotide, and (x) ligating the nucleic acid strands to the further identifier oligonucleotides in the double-stranded complexes, such that the further coding sequence identifier oligonucleotides are incorporated into the nucleic acid strands.
 50. A method according to claim 49 comprising cleaving the further adaptor to remove it from the nucleic acid strands and the further identifier oligonucleotides, optionally wherein the further adaptor comprises one or more ribonucleotide bases.
 51. A method of producing a nucleic acid encoded chemical library comprising; (i) providing a sub-library of first nucleic acid strands coupled to first and second diverse populations of chemical moieties (“first and second chemical moieties”), wherein each first nucleic acid strand comprises a first coding sequence which encodes the member of the first diverse population of chemical moieties that is coupled to the first nucleic acid strand, (ii) contacting the first nucleic acid strands with an adaptor oligonucleotide and first identifier oligonucleotides comprising coding sequences, such that the adaptor oligonucleotide hybridizes to the first nucleic acid strands and the first identifier oligonucleotides to form partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a first identifier oligonucleotide comprising a coding sequence which encodes the member of the second population of chemical moieties that is coupled to the first nucleic acid strand, and; wherein all the first nucleic acid strands in the sub-library are contacted with the same adaptor oligonucleotide, (iii) ligating the first nucleic acid strands to the first identifier oligonucleotides in the complexes, such that the second coding sequences are incorporated into the first nucleic acid strands; (iv) contacting the first nucleic acid strands with a nucleic acid spacer strand, second identifier oligonucleotides, and a sub-library of second nucleic acid strands coupled to a third diverse population of chemical moieties (“third chemical moieties”), thereby forming partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a second identifier oligonucleotide comprising a third coding sequence that encodes the member of the third population of chemical moieties that is coupled to the second nucleic acid strand, and; wherein all the nucleic acid strands in the population are contacted with the same nucleic acid spacer strand, (v) ligating the first nucleic acid strand to the second identifier oligonucleotide such that the third coding sequence is incorporated into the nucleic acid strand; and, (vi) optionally ligating the second nucleic acid strand to the nucleic acid spacer strand, thereby producing a library comprising pharmacophores labelled with double-stranded nucleic acid molecules comprising first and second nucleic acid strands, wherein the nucleic acid spacer strand comprises; a) a first hybridization portion which hybridizes to the first nucleic acid strand, b) a non-hybridizable spacer at a position that corresponds, when the first nucleic acid strand and the nucleic acid spacer strand are hybridised together, to the position of the second coding sequence in the first nucleic acid strand c) a second hybridization portion which hybridizes to the first nucleic acid strand; and, d) a complementary annealing region which hybridizes to the second identifier oligonucleotide.
 52. A method according to claim 51 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 53. A method according to claim 51 wherein the adaptor oligonucleotide is removed after the ligation by cleaving the adaptor oligonucleotide.
 54. A method according to claim 53 wherein the adaptor oligonucleotide comprises one or more ribonucleotide bases.
 55. A method of producing a nucleic acid encoded chemical library comprising; (i) providing a sub-library of first nucleic acid strands coupled to first and second diverse populations of chemical moieties (“first and second chemical moieties”), wherein each first nucleic acid strand comprises a first coding sequence which encodes the member of the first diverse population of chemical moieties that is coupled to the first nucleic acid strand, (ii) contacting the first nucleic acid strands with an adaptor oligonucleotide and first identifier oligonucleotides comprising coding sequences, such that the adaptor oligonucleotide hybridizes to the first nucleic acid strands and the first identifier oligonucleotides to form partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a first identifier oligonucleotide comprising a coding sequence which encodes the member of the second population of chemical moieties that is coupled to the first nucleic acid strand, and; wherein each adaptor oligonucleotide hybridizes to more than one nucleic acid strand in the population and more than one different identifier oligonucleotide, (iii) ligating the first nucleic acid strands to the first identifier oligonucleotides in the complexes, such that the second coding sequences are incorporated into the first nucleic acid strands; (iv) contacting the first nucleic acid strands with one or more nucleic acid spacer strands, second identifier oligonucleotides, and a sub-library of second nucleic acid strands coupled to a third diverse population of chemical moieties (“third chemical moieties”), thereby forming partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a second identifier oligonucleotide comprising a third coding sequence that encodes the member of the third population of chemical moieties that is coupled to the second nucleic acid strand, and wherein the second nucleic acid strands, nucleic spacer strand and second identifier oligonucleotides hybridise to the first nucleic acid strand to form a double-stranded complex having a 5′ overhang comprising the third coding sequence; wherein each spacer strand hybridizes to more than one first nucleic acid strand in the population and more than one different second identifier oligonucleotide, (v) extending the first nucleic acid strand along the second identifier oligonucleotide to incorporate the complement of the third coding sequence into the first nucleic acid strand; and, (vi) optionally ligating the second nucleic acid strands to the nucleic acid spacer strands and the second identifier oligonucleotides, thereby producing a library comprising pharmacophores labelled with double-stranded nucleic acid molecules comprising first and second nucleic acid strands, wherein the nucleic acid spacer strand comprises; a) a first hybridization portion which hybridizes to the first nucleic acid strand, b) a non-hybridizable spacer at a position that corresponds, when the first nucleic acid strand and the nucleic acid spacer strand are hybridised together, to the position of the second coding sequence in the first nucleic acid strand c) a second hybridization portion which hybridizes to the first nucleic acid strand.
 56. A method according to claim 55 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 57. A method according to claim 55 wherein the adaptor oligonucleotide is removed after the ligation by cleaving the adaptor oligonucleotide.
 58. A method according to claim 57 wherein the adaptor oligonucleotide comprises one or more ribonucleotide bases.
 59. A method of producing a nucleic acid encoded chemical library comprising; (i) providing a sub-library of first nucleic acid strands coupled to first and second diverse populations of chemical moieties (“first and second chemical moieties”), wherein each first nucleic acid strand comprises a first coding sequence which encodes the member of the first diverse population of chemical moieties that is coupled to the first nucleic acid strand, (ii) contacting the first nucleic acid strands with an adaptor oligonucleotide and first identifier oligonucleotides comprising coding sequences, such that the adaptor oligonucleotide hybridizes to the first nucleic acid strands and the first identifier oligonucleotides to form partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a first identifier oligonucleotide comprising a coding sequence which encodes the member of the second population of chemical moieties that is coupled to the first nucleic acid strand, and; wherein each adaptor oligonucleotide hybridizes to more than one nucleic acid strand in the population and more than one different identifier oligonucleotide, (iii) ligating the first nucleic acid strands to the first identifier oligonucleotides in the complexes, such that the second coding sequences are incorporated into the first nucleic acid strands; (iv) contacting the first nucleic acid strands with a sub-library of second nucleic acid strands coupled to a third diverse population of chemical moieties (“third chemical moieties”), thereby forming partially double-stranded complexes, wherein each second nucleic acid strand comprises first and second non-hybridizable spacer regions at positions corresponding to the first and second coding sequences in the first nucleic acid strand and a third coding sequence that encodes the member of the third population of chemical moieties that is coupled to the second nucleic acid strand, and wherein the second nucleic acid strands hybridise to the first nucleic acid strand to form a double-stranded complex having a 5′ overhang comprising the third coding sequence; (v) extending the first nucleic acid strand along the second identifier oligonucleotide to incorporate the complement of the third coding sequence into the first nucleic acid strand; thereby producing a library comprising pharmacophores labelled with double-stranded nucleic acid molecules comprising first and second nucleic acid strands.
 60. A method according to claim 59 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 61. A method according to claim 59 wherein the adaptor oligonucleotide is removed after the ligation by cleaving the adaptor oligonucleotide.
 62. A method according to claim 61 wherein the adaptor oligonucleotide comprises one or more ribonucleotide bases
 63. A method according to claim 59 wherein the sub-library of second nucleic acid strands is produced by a method comprising; (a) providing a second nucleic acid strand having a third chemical moiety coupled thereto, wherein the second nucleic acid strand comprises a first non-hybridizable spacer region at a position corresponding to the first coding sequence in the first nucleic acid strand, (b) contacting the second nucleic acid strand with an adaptor oligonucleotide and a nucleic acid spacer strand comprising a second non-hybridizable spacer region at a position corresponding to the second coding sequence in the first nucleic acid strand, such that the adaptor oligonucleotide hybridizes to the second nucleic acid strand and the nucleic acid spacer strand to form a partially double-stranded complex, (c) ligating the second nucleic acid strand to the nucleic acid spacer strand in the complex, such that the second non-hybridizable spacer region is incorporated into the second nucleic acid strand; (d) contacting the second nucleic acid strand with an adaptor oligonucleotide and a second identifier oligonucleotide comprising a third coding sequence that encodes the third chemical moiety, such that the adaptor oligonucleotide hybridizes to the second nucleic acid strand and the second identifier oligonucleotide to form a partially double-stranded complex, (e) ligating the second nucleic acid strand to the second identifier oligonucleotide in the complex, such that the third coding sequence is incorporated into the second nucleic acid strand; (f) repeating steps (a) to (e) in series or in parallel using different third chemical moieties and third coding sequences and the same adaptor oligonucleotide to produce a diverse population of third chemical moieties coupled to second nucleic acid strands, each third chemical moiety being coupled to a second nucleic acid strand which comprises first and second spacer regions and a third coding sequence encoding the third chemical moiety coupled thereto.
 64. A method according to claim 63 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 65. A method according to claim 63 wherein the adaptor oligonucleotide is removed after the ligation by cleaving the adaptor oligonucleotide.
 66. A method according to claim 65 wherein the adaptor oligonucleotide comprises one or more ribonucleotide bases.
 67. A method of producing a nucleic acid encoded chemical library comprising; (i) providing a sub-library of first nucleic acid strands coupled to first and second diverse populations of chemical moieties (“first and second chemical moieties”), wherein each first nucleic acid strand comprises a first coding sequence which encodes the member of the first diverse population of chemical moieties that is coupled to the first nucleic acid strand, (ii) contacting the first nucleic acid strands with one or more nucleic acid spacer strands and first identifier oligonucleotides comprising coding sequences, such that the first nucleic acid spacer strand hybridizes to the first nucleic acid strands and the first identifier oligonucleotides to form partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a first identifier oligonucleotide comprising a second coding sequence which encodes the member of the second population of chemical moieties that is coupled to the first nucleic acid strand, and; wherein each nucleic acid spacer strand hybridizes to more than one first nucleic acid strand in the population and more than one different first identifier oligonucleotide, (iii) ligating the first nucleic acid strands to the first identifier oligonucleotides in the complexes, such that the second coding sequences are incorporated into the first nucleic acid strands; (iv) contacting the first nucleic acid strands hybridised to the nucleic acid spacer strand with a sub-library of second nucleic acid strands coupled to a third diverse population of chemical moieties (“third chemical moieties”) and second identifier oligonucleotides, thereby forming double-stranded complexes, wherein each first nucleic acid strand is contacted with a second identifier oligonucleotide that comprises a third coding sequence that encodes the member of the third population of chemical moieties that is coupled to the second nucleic acid strand that is contacted therewith, and; (v) ligating the first nucleic acid strand to the second identifier oligonucleotide such that the third coding sequence is incorporated into the nucleic acid strand; and, (vi) optionally ligating the second nucleic acid strand to the nucleic acid spacer strand, thereby producing a library comprising pharmacophores labelled with double-stranded nucleic acid molecules comprising first and second nucleic acid strands, wherein the nucleic acid spacer strand comprises; a) a first hybridization portion which hybridizes to the first nucleic acid strand, b) a first non-hybridizable spacer at a position that corresponds, when the first nucleic acid strand and the nucleic acid spacer strand are hybridised together, to the position of the second coding sequence in the first nucleic acid strand, c) a second hybridization portion which hybridizes to the first nucleic acid strand; d) a first annealing region which hybridizes to the second identifier oligonucleotide, e) a second non-hybridizable spacer at a position that corresponds, when the second identifier oligonucleotide and the nucleic acid spacer strand are hybridised together, to the position of the third coding sequence in the second identifier oligonucleotide and; f) a second annealing region which hybridizes to the second identifier oligonucleotide.
 68. A method according to claim 67 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 69. A method of producing a nucleic acid encoded chemical library comprising; (i) providing a sub-library of first nucleic acid strands coupled to first and second diverse populations of chemical moieties (“first and second chemical moieties”), wherein each first nucleic acid strand comprises a first coding sequence which encodes the member of the first diverse population of chemical moieties that is coupled to the first nucleic acid strand, (ii) contacting the first nucleic acid strands with one or more nucleic acid spacer strands and first identifier oligonucleotides comprising coding sequences, such that the first nucleic acid spacer strand hybridizes to the first nucleic acid strands and the first identifier oligonucleotides to form partially double-stranded complexes, wherein each first nucleic acid strand is contacted with a first identifier oligonucleotide comprising a second coding sequence which encodes the member of the second population of chemical moieties that is coupled to the first nucleic acid strand, and; wherein each nucleic acid spacer strand hybridizes to more than one first nucleic acid strand in the population and more than one different first identifier oligonucleotide, (iii) ligating the first nucleic acid strands to the first identifier oligonucleotides in the complexes, such that the second coding sequences are incorporated into the first nucleic acid strands; (iv) contacting the first nucleic acid strands with second identifier oligonucleotides and a sub-library of second nucleic acid strands coupled to a third diverse population of chemical moieties (“third chemical moieties”), thereby forming double-stranded complexes, wherein each first nucleic acid strand is contacted with a second identifier oligonucleotide comprising a third coding sequence that encodes the member of the third population of chemical moieties that is coupled to the second nucleic acid strand, and; the double-stranded complexes have a 5′ overhang comprising the third coding sequence, (v) extending the first nucleic acid strand along the second nucleic acid strand to incorporate the complement of the third coding sequence into the first nucleic acid strand; and, thereby producing a library comprising pharmacophores labelled with double-stranded nucleic acid molecules comprising first and second nucleic acid strands, wherein the second nucleic acid strand comprises; a) a first hybridization portion which hybridizes to the first nucleic acid strand, b) a non-hybridizable spacer at a position that corresponds, when the first and second strands are hybridised together, to the position of the first coding sequence in the first nucleic acid strand; and c) a second hybridization portion which hybridizes to the first nucleic acid strand.
 70. A method according to claim 69 wherein the spacer comprises an abasic linker, optionally an abasic deoxyribose phosphate linker.
 71. A nucleic acid encoded chemical library produced by the method of claim
 43. 72. A method of screening a nucleic acid encoded chemical library comprising; producing a nucleic acid encoded chemical library by the method of claim 43, contacting the library with a target molecule and selecting one or more library members which bind to the target, optionally wherein the method further comprises; (i) isolating the nucleic acid strand of the library members, (ii) amplifying the nucleic acid strand of the selected library members, and/or (iii) determining the sequence of the nucleic acid strands of the selected library members to identify the chemical moieties of the selected library members. 